Histone acetyltransferase activators and compositions and uses thereof

ABSTRACT

The invention provides pharmaceutical compositions and methods for treating cancer, neurodegenerative disorders, conditions associated with accumulated amyloid-beta peptide deposits, Tau protein levels, and/or accumulations of alpha-synuclein by administering a HAT modulator and a HDAC modulator to a subject.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.15/655,583, filed on Jul. 20, 2017, which claims the benefit of U.S.Provisional Patent Application No. 62/364,480, filed on Jul. 20, 2016and entitled “Histone Acetyltransferase Activators and Uses Thereof,”the disclosure of which is hereby incorporated by reference in itsentirety for all purposes.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Modulation of the acetylation state of histones, transcription factors,and other regulatory proteins is known to influence their activitywithin cancer and inflammatory cells. The acetylation state of a proteinis controlled by the activity of two main groups of enzymes, histonedeacetylases (HDAC) and histone acetyl transferases (HAT). The HDACremoves acetyl-groups while the HATs transfer acetyl-groups to theprotein of interest.

Classically, modulation of acetylation status is known to influence thecondensation of chromatin. In cancer, histones are deacetylatedmaintaining a condensed chromatin structure, and a transcriptionallysilenced state. This transcriptional inactivation is mediated by HDACswhich remove acetyl groups from histone tails, maintain a condensedchromatic structure. Inhibitors of HDACs help maintain transcriptionallyactive chromatin, theoretically allowing for expression of tumorsuppressor genes. One observation that has evolved is that histones arenot the only targets of acetylation. It is now accepted thatpost-translational acetylation of intracellular proteins such as tumorsuppressors (p53) and oncogenes (Bcl6) plays a critical role ininfluencing their activity. It has been established that there is anetwork of proteins and enzymes that can be modified by acetylation, nowcollectively referred to as the acetylome.

Cognitive neurodegenerative disorders are characterized by synapticdysfunction, cognitive abnormalities, and/or the presence of inclusionbodies throughout the CNS containing, for example, but not limited tonative beta-amyloid fragments, native and phosphorylated Tau, native andphosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TDB-43), invarious percentages and in relation to the specific disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative diseasecharacterized by memory loss, synaptic dysfunction and accumulation ofamyloid β-peptides (Aβ). The pathogenesis of AD is believed to be causedby high levels and aggregation of amyloid-β (Aβ) in the brain. Aβ hasbeen found to impair memory by reducing acetylation of specific histonelysines important for memory formation. Histones are proteins thatclosely associate with DNA molecules and play an important role in genetranscription.

Currently available therapies for AD are palliative and do not cure thedisease. Cholinesterase inhibitors such as Razadyne® (galantamine),Exelon® (rivastigmine), Aricept® (donepezil), and Cognex® (tacrine) havebeen prescribed for early stages of Alzheimer's disease, and maytemporarily delay or prevent progression of symptoms related to AD.However, as AD progresses, the brain loses less acetylcholine, therebyrendering cholinesterase inhibitors unproductive as treatment for AD.Namenda® (memantine), an N-methyl D-aspartate (NMDA) antagonist, is alsoprescribed to treat moderate to severe Alzheimer's disease; however onlytemporary benefits are realized.

Histone Acetyltransferases (HATs) are involved in histone acetylation(leading to gene activation), chromosome decondensation, DNA repair andnon-histone substrate modification. The post-translational acetylationstatus of chromatin is governed by the competing activities of twoclasses of enzymes, HATs and HDACs. The potential of inhibiting HDACs tocounteract neurodegenerative disorders has been widely explored (CurrDrug Targets CNS Neurol Disord, 2005. 4(1): p. 41-50; herebyincorporated by reference in its entirety). HATs, however, have beeninvestigated to a lesser extent. HAT activators have been reported, butmany are neither soluble nor membrane permeant, which makes them poorcandidates for therapeutics. CTPB and CTB are HAT activators that areinsoluble and membrane-impermeable (J Phys Chem B, 2007. 111(17): p.4527-34; J Biol Chem, 2003. 278(21): p. 19134-40; each herebyincorporated by reference in its entirety). Nemorosone is another HATactivator (Chembiochem. 11(6): p. 818-27; hereby incorporated byreference in its entirety). However, these compounds suffer fromunfavorable physicochemical characteristics for use in CNS diseases.

There is a need for novel HAT activators. There is also a need for noveltreatments for a variety of disease states for which HAT activity isimplicated. There is a further need for novel and effective treatmentsfor neurodegenerative diseases, neurological disorders and cancers. Inparticular, there is a continuing need for treatment of dementia andmemory loss associated with Alzheimer's disease. There is also acontinuing need for treatment of cancer.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to pharmaceutical compositionsand methods for treating cancer in a subject in need thereof. Thepharmaceutical compositions may comprise a HAT activator and a HDACinhibitor and the methods may comprise administering to a subject a HATactivator and a HDAC inhibitor.

In one embodiment of the pharmaceutical compositions disclosed herein,the HAT activator has a structure of formula (I),

wherein,

Ar is

R^(a) is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;

R^(b) is H, OH, halogen, C₁-C₆-alkyl, —(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl),C₁-C₆-haloalkyl, C₂-C₆-alkenyl, C₃-C₈-cycloalkyl, C₂-C₆-heteroalkyl,C₃-C₈-heterocycloalkyl, aryl, heteroaryl, O—(C₁-C₆-alkyl),O—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), O—(C₁-C₆-haloalkyl),O—(C₃-C₈-cycloalkyl), O—(C₂-C₆-alkenyl), O—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₁-C₆-alkyl), N(R¹⁰)—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl),N(R¹⁰)—(C₃-C₈-cycloalkyl), SH, S—(C₁-C₆-alkyl),S—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), SO₂—(C₁-C₆-alkyl),SO₂—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)-N(R¹⁰)₂,O—(C₂-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻, O—(C₃-C₈-cycloalkyl)-N(R¹⁰)₂,N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-R³,O—(C₁-C₆-alkyl)-R³, O—(C₃-C₈-cycloalkyl)-R³, N(R¹⁰)—(C₁-C₆-alkyl)-R³,O-aryl, or O-heteroaryl;

R^(c) is H, —(C₁-C₆-alkyl), O—(C₁-C₆-alkyl), C(═O)NH-phenyl, whereinphenyl is substituted with one or more halo or haloalkyl;

R^(d) is H, OH, halogen, C₁-C₁₆-alkyl, C₁-C₁₆-haloalkyl,O—(C₃-C₈-cycloalkyl), O—(C₃-C₈-heterocycloalkyl), O—(C₂-C₆-alkenyl),O—(C₁-C₆-alkyl), O—(C₁-C₆-alkyl)-phenyl, O—(C₂-C₆-alkyl)-N(R¹⁰)₂,O—(C₂-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻, —(C₁-C₆-alkyl)-R³,O—(C₁-C₆-alkyl)-R³, O—S(C₁-C₆-alkyl), N(R¹⁰)—(C₁-C₆-alkyl)-R³,—N(R¹⁰)—(C₁-C₆-alkyl), —N(R¹⁰)—(C₂-C₆-alkenyl),—N(R¹⁰)—(C₃-C₈-cycloalkyl), —N(R¹⁰)—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-N(R¹⁰)₂,S—(C₂-C₆-alkyl)-N(R¹⁰)₂, OCH₂C(O)O(C₁-C₆-alkyl), O-aryl, N-aryl,O-heteroaryl, or N-heteroaryl;

U¹-U⁴ are independently N or CR^(a), wherein U¹-U⁴ are not each N;

V is a bond, N or CR^(c);

W and Z are independently N or CR¹;

X is —CO—, —CON(R¹⁰)—, —CON(R¹⁰)(CH₂)_(n)—, —(CH₂)_(n)CON(R¹⁰)—,—(CH₂)_(n)CON(R¹⁰)(CH₂)_(n)—, —SON(R¹⁰)—, —SON(R¹⁰)(CH₂)_(n)—,—SO₂N(R¹⁰)—, —SO₂N(R¹⁰)(CH₂)_(n)—, —N(R¹⁰)C(═O)N(R¹⁰)—, —N(R¹⁰)CO—,—N(R¹⁰)CO(CH₂)_(n)—, or —N(R¹⁰)CO(CH₂)_(n)—, —(CH₂)_(n)N(R¹⁰)—, —C═N—;or

Ar and X together form

Y is a bond, N or CR²;

R¹ is H, halogen, O—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)N(R¹⁰)₂;

R² is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;

R³ is cycloalkylamino, optionally containing a heteroatom selected fromN(R¹⁰), O and S;

R¹⁰ is independently H, —(C₁-C₄-alkyl), —(C₁-C₄-haloalkyl),—(C₃-C₈-cycloalkyl), —(C₃-C₈-heterocycloalkyl), aryl or heteroaryl;

-   -   is a double bond and R¹¹ is O; or    -   is a single bond and R¹¹ is —(C₁-C₆-alkyl),        —(C₁-C₆-alkyl)-N(R¹⁰)₂, or —(C₁-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen; and        -   each n is independently an integer from 1-4, or a            pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical compositions disclosed herein,the HAT activator is selected from the group consisting of:

wherein R is H, Methyl, Ethyl, n-Propyl, Isopropyl, n-butyl, t-butyl,C₈H₁₈, C₁₅H₂₆, C₁₅H₂₈, C₁₅H₃₀, or C₁₅H₃₂.

In one embodiment of the pharmaceutical compositions disclosed herein,the HAT activator is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical compositions disclosed herein,the HAT activator is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

In one embodiment of the pharmaceutical compositions disclosed herein,the HAT activator is

or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.

In one embodiment of the pharmaceutical compositions disclosed herein,the HAT activator is

or a pharmaceutically acceptable salt thereof and a HDAC inhibitor. In aspecific embodiment, the HAT activator is

or a pharmaceutically acceptable salt thereof, and the HDAC inhibitor isromidepsin.

In one aspect, the invention is directed to methods for treating cancerin a subject in need thereof, the method comprising administering thepharmaceutical compositions above to a subject. In one embodiment of theany one of the methods disclosed herein, the cancer comprises B celllymphoma, colon cancer, lung cancer, renal cancer, bladder cancer, Tcell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, acute lymphocyticleukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lungcancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma,uterine cancer, renal cell carcinoma, hepatoma, adenocarcinoma, breastcancer, pancreatic cancer, liver cancer, prostate cancer, head and neckcarcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer,primary or metastatic melanoma, squamous cell carcinoma, basal cellcarcinoma, brain cancer, angiosarcoma, hemangiosarcoma, bone sarcoma,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, testicular cancer, uterinecancer, cervical cancer, gastrointestinal cancer, mesothelioma, Ewing'stumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, Waldenstroom'smacroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma,bronchogenic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, lung carcinoma, epithelial carcinoma,cervical cancer, testicular tumor, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, retinoblastoma, leukemia,melanoma, neuroblastoma, small cell lung carcinoma, bladder carcinoma,multiple myeloma, follicular lymphoma or medullary carcinoma.

In one embodiment of the any one of the methods disclosed herein, theHAT activator increases histone acetylation. In another embodiment ofthe any one of the methods disclosed herein, the HDAC inhibitorincreases histone acetylation. In some embodiments, histone acetylationcomprises acetylation of histones H2B, H3, H4, or a combination thereof.In other embodiments, histone acetylation comprises acetylation ofhistone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, ora combination thereof.

In one embodiment of the any one of the methods disclosed herein, theHAT activator increases p53 acetylation. In another embodiment of theany one of the methods disclosed herein, the HDAC inhibitor increasesp53 acetylation.

In one embodiment of the any one of the methods disclosed herein, theIDAC inhibitor is romidepsin, vorinostat, belinostat, panobinostat,entinostat, mocetinostat, abexinostat, quisinostat or gavinostat. Insome embodiments, the HDAC inhibitor is romidepsin or vorinostat. Inanother embodiment, the HDAC inhibitor is romidepsin.

In one embodiment of the any one of the methods disclosed herein, thecancer is colon cancer, lung cancer, renal cancer, leukemia, CNS cancer,melanoma, ovarian cancer, breast cancer, or prostate cancer. In otherembodiments, the cancer is colon cancer, renal cancer, T cell leukemia,myeloma, leukemia, acute myeloid leukemia, acute lymphocytic leukemia,renal cell carcinoma, adenocarcinoma, glioblastoma, breast carcinoma,prostate carcinoma, or lung carcinoma. In one embodiment, the cancer isHodgkin's lymphoma, non-Hodgkin's lymphoma, B cell lymphoma, T celllymphoma, or follicular lymphoma. In some embodiments, the B celllymphoma is diffuse large B-cell lymphoma.

In one embodiment of the any one of the methods disclosed herein, thecancer is diffuse large B-cell lymphoma selected from germinalcenter-derived diffuse large B cell lymphoma, an activatedB-cell-derived (ABC) diffuse large B-cell lymphoma, or non-germinalcenter diffuse large B cell lymphoma.

Another aspect of the invention provides a method for reducing amyloidbeta (A) protein deposits in a subject in need thereof, the methodcomprising administering to the subject a HAT activator and a HDACinhibitor.

In one embodiment of the anyone of the methods disclosed herein, thesubject exhibits abnormally elevated levels of amyloid beta plaques. Inanother embodiment, the subject is afflicted with Alzheimer's disease,Lewy body dementia, inclusion body myositis, or cerebral amyloidangiopathy. Another aspect of the invention provides a method fortreating a neurodegenerative disease in a subject, the method comprisingadministering to a subject a HAT activator and a HDAC inhibitor.

In one embodiment of the any one of the methods disclosed herein, theneurodegenerative disease comprises Adrenoleukodystrophy (ALD),Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease,Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Rett's syndrome, Tau-positive FrontoTemporaldementia, Tau-negative FrontoTemporal dementia, Refsum's disease,Sandhoff disease, Schilder's disease, Subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease, Batten disease, Spinocerebellarataxia, Spinal muscular atrophy, Steele-Richardson-Olszewski disease,Tabes dorsalis, or Toxic encephalopathy. In another embodiment, theneurodegenerative disease is selected from Alzheimer's Disease, ALS,Parkinson's Disease, and Huntington's Disease. In some embodiments, theneurodegenerative disease is Alzheimer's Disease. In other embodiments,the neurodegenerative disease is Huntington's Disease.

Another aspect of the invention provides a method for increasing memoryretention in a subject afflicted with a neurodegenerative disease, themethod comprising administering to a subject a HAT activator and a HDACinhibitor.

In one embodiment of the any one of the methods disclosed herein, theneurodegenerative disease comprises Adrenoleukodystrophy (ALD),Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease,Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Rett's syndrome, Tau-positive FrontoTemporaldementia, Tau-negative FrontoTemporal dementia, Refsum's disease,Sandhoff disease, Schilder's disease, Subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease, Batten disease, Spinocerebellarataxia, Spinal muscular atrophy, Steele-Richardson-Olszewski disease,Tabes dorsalis, or Toxic encephalopathy. In another embodiment, theneurodegenerative disease is Alzheimer's Disease.

In one aspect, the invention is directed to a method for treating cancerin a subject in need thereof, the method comprising administering to asubject

or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.

Another aspect of the invention provides a method for treating cancer ina subject in need thereof, the method comprising administering to asubject

or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows chemical structures of representative HAT modulatorcompounds.

FIG. 2 shows scheme of synthesis of RP52.

FIG. 3 shows scheme of synthesis of RP14, RP58, and RP59.

FIG. 4 shows scheme of synthesis of JF2.

FIGS. 5A-C show synergy between romidepsin and RP14 evaluated byluminetric assays in cell line Ly7. Cytotoxicity was measured at 24 (A),48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculated forthe Romidepsin in combination with RP14 treatment and is shown above thebars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR<1 represents anantagonistic interaction, RRR=1 additive, and RRR>1 synergisticinteraction.

FIGS. 6A-C show synergy between romidepsin and RP14 evaluated byluminetric assays in cell line Ly10. Cytotoxicity was measured at 24(A), 48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculatedfor the Romidepsin in combination with RP14 treatment and is shown abovethe bars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 representsan antagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 7A-C show synergy between romidepsin and RP14 evaluated byluminetric assays in cell line SuDHL-6. Cytotoxicity was measured at 24(A), 48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculatedfor the Romidepsin in combination with RP14 treatment and is shown abovethe bars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 representsan antagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 8A-C show synergy between romidepsin and RP52 evaluated byluminetric assays in cell line Ly7. Cytotoxicity was measured at 24 (A),48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculated forthe Romidepsin in combination with RP52 treatment and is shown above thebars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 represents anantagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 9A-C show synergy between romidepsin and RP52 evaluated byluminetric assays in cell line Ly10. Cytotoxicity was measured at 24(A), 48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculatedfor the Romidepsin in combination with RP52 treatment and is shown abovethe bars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 representsan antagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 10A-C show synergy between romidepsin and RP52 evaluated byluminetric assays in cell line SuDHL-6. Cytotoxicity was measured at 24(A), 48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculatedfor the Romidepsin in combination with RP52 treatment and is shown abovethe bars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 representsan antagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 11A-C show synergy between romidepsin and RP59 evaluated byluminetric assays in cell line Ly7. Cytotoxicity was measured at 24 (A),48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculated forthe Romidepsin in combination with RP59 treatment and is shown above thebars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 represents anantagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 12A-C show synergy between romidepsin and RP59 evaluated byluminetric assays in cell line Ly10. Cytotoxicity was measured at 24(A), 48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculatedfor the Romidepsin in combination with RP59 treatment and is shown abovethe bars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 representsan antagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 13A-C show synergy between romidepsin and RP59 evaluated byluminetric assays in cell line SuDHL-6. Cytotoxicity was measured at 24(A), 48 (B) and 72 (C) hours. Synergy coefficient (RRR) was calculatedfor the Romidepsin in combination with RP59 treatment and is shown abovethe bars labeled R+1, R+2, R+4, R+6, R+8, or R+10. An RRR>1 representsan antagonistic interaction, RRR=1 additive, and RRR<1 synergisticinteraction.

FIGS. 14A-C show synergy between romidepsin and different HAT activators(RP14, RP52, RP72, JF2) evaluated by luminetric assays in cell line H9.Cytotoxicity was measured at 24 (A), 48 (B) and 72 (C) hours.

FIGS. 15A-B show synergy between romidepsin and different HAT activators(RP14, RP52, RP72, JF2) evaluated by luminetric assays in cell line HH.Cytotoxicity was measured at 48 (A), and 72 (C) hours.

FIGS. 16A-C show synergy between romidepsin and RP14 evaluated byluminetric assays in cell line H9. Cytotoxicity was measured at 24 (A),48 (B) and 72 (C) hours.

FIGS. 17A-C show synergy between romidepsin and RP14 evaluated byluminetric assays in cell line HH. Cytotoxicity was measured at 24 (A),48 (B) and 72 (C) hours.

FIGS. 18A-C show synergy between romidepsin and RP72 evaluated byluminetric assays in cell line HH. Cytotoxicity was measured at 24 (A),48 (B) and 72 (C) hours.

FIGS. 19A-B show acetylation of p53 and modulation of p21 by HATactivator RP52 in diffuse large B-cell lymphoma cell lines Ly1 (A) andSu-DHL6 (B).

FIGS. 20A-D show the concentration-effect relationship for 21 HATactivator compounds in a panel of non-Hodgkin's lymphoma cell lines at48 hours. FIG. 20A shows the percentage viability of cells treated withHAT activators YF2, JF1, JF3, JF4, JF5, JF7, JF8, JF9, JF10, JF16, JF18as single agents or in combination with romidepsin at 48 hours. FIG. 20Bshows the percentage viability of cells treated with HAT activatorsRP14, RP17, RP23, RP52, RP58, RP59, RP72, RP78, RP79, RP102 as singleagents or in combination with romidepsin at 48 hours. FIG. 20C shows thesynergy coefficients calculated as the relative risk ratio (RRR) forcells treated with HAT activators YF2, JF1, JF3, JF4, JF5, JF7, JF8,JF9, JF10, JF16, JF18 as single agents or in combination with romidepsinat 48 hours. FIG. 20D shows the synergy coefficients calculated as therelative risk ratio (RRR) for cells treated with HAT activators RP14,RP17, RP23, RP52, RP58, RP59, RP72, RP78, RP79, RP102 as single agentsor in combination with romidepsin at 48 hours.

FIG. 21 shows in vitro measurement of pCAF activity for compounds.

FIGS. 22A-E show the synergy effect of JF1 with romidepsin in B-celllymphoma cell lines. Pfeiffer cells (FIG. 22A) and SUDHL-10 cells (FIG.22B) were exposed to increasing concentration of JF1 and romidepsinalone and in combinations at 72 hrs. Five B-cell lymphoma cell lineswere exposed to increasing concentration of JF1 and romidepsin alone andin combinations at 48 hrs (FIG. 22C) and 72 hrs (FIG. 22D) and theExcess Over Bliss was measured for each. Synergy is defined by an ExcessOver Bliss of 10. The synergy of JF1 and romidepsin in each cell lineand at each time point is further indicated in FIG. 22E.

FIG. 23 shows the synergy effect of increasing concentrations of JF1 andromidepsin in ten B-cell lymphoma cell lines and four T-cell lymphomacell lines alone and in combination at 72 hrs. Strong synergy is definedby an Excess Over Bliss of 20.

FIG. 24 shows the synergy effect of increasing concentrations of YF2 andromidepsin in seven B-cell lymphoma cell lines alone and in combinationat 72 hrs. Strong synergy is defined by an Excess Over Bliss of 20.

FIGS. 25A-B compare the synergistic effects of JF1 and YF2 on histoneacetylation in B-cell lymphoma cell lines alone and in combination at 48hrs, as quantified by mass spectrometry. The fold changes vs. controlfor histone acetylation in SUDHL-6 (FIG. 25A) and SUDHL-10 (FIG. 25B)were calculated for five lysine-acetylated histones.

FIG. 26 provides a Western Blot showing the effect of JF1 and YF2 aloneand in combination with romidepsin on histone acetylation in HAT-mutatedcells lines, SUDHL-6 and SUDHL-10. Cells were exposed to YF2 andromidepsin alone and in combinations for 48 hours in SUDHL-6 andSUDHL-10 (EP300 mut/CREBBP mut).

FIG. 27 shows the synergy of JF1/YF2 and romidepsin in each cell line at72 hr with EOB, which provides further details for FIG. 23 and FIG. 24.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

An “effective amount”, “sufficient amount” or “therapeutically effectiveamount” as used herein is an amount of a compound that is sufficient toeffect beneficial or desired results, including clinical results. Assuch, the effective amount may be sufficient, for example, to reduce orameliorate the severity and/or duration of an affliction or condition,or one or more symptoms thereof, prevent the advancement of conditionsrelated to an affliction or condition, prevent the recurrence,development, or onset of one or more symptoms associated with anaffliction or condition, or enhance or otherwise improve theprophylactic or therapeutic effect(s) of another therapy. An effectiveamount also includes the amount of the compound that avoids orsubstantially attenuates undesirable side effects.

As used herein and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results may include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminution of extent of disease, a stabilized (i.e., notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

The term “in need thereof” refers to the need for symptomatic orasymptomatic relief from a condition such as, for example, cancer or aneurodegenerative disease. The subject in need thereof may or may not beundergoing treatment for conditions related to, for example, cancer or aneurodegenerative disease.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Non-limiting examples of suchpharmaceutical carriers include liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical carriers may also be saline, gum acacia, gelatin,starch paste, talc, keratin, colloidal silica, urea, and the like. Inaddition, auxiliary, stabilizing, thickening, lubricating and coloringagents may be used. Other examples of suitable pharmaceutical carriersare described in Remington: The Science and Practice of Pharmacy,21^(st) Edition (University of the Sciences in Philadelphia, ed.,Lippincott Williams & Wilkins 2005); and Handbook of PharmaceuticalExcipients, 7^(th) Edition (Raymond Rowe et al., ed., PharmaceuticalPress 2012); each hereby incorporated by reference in its entirety.

The terms “animal,” “subject” and “patient” as used herein includes allmembers of the animal kingdom including, but not limited to, mammals,animals (e.g., cats, dogs, horses, swine, etc.) and humans.

In some embodiments, the invention is directed to the combination of oneor more HAT modulator compounds and one or more HDAC modulatorcompounds. In some embodiments, the HAT modulator is a HAT activator. Insome embodiments, the HAT modulator is a HAT inhibitor. In otherembodiments, the HDAC modulator is a HDAC inhibitor. In otherembodiments, the HDAC inhibitor is a HDAC activator. In someembodiments, the invention is directed to the combination of one or moreHAT activators and one or more HDAC inhibitors. In some embodiments, theinvention is directed to the combination of a HAT activator and a HDACinhibitor. In other embodiments, the combination of a HAT activator anda HDAC inhibitor is unexpectedly effective for killing cancer cells. Inother embodiments, the combination of a HAT activator and a HDACinhibitor is unexpectedly cytotoxic to cancer cells. In someembodiments, the HAT activator increases histone acetylation. In someembodiments, the HDAC inhibitor increases histone acetylation. In someembodiments, the histone acetylation comprises acetylation of histonesH2B, H3, H4, or a combination thereof. In some embodiments, the histoneacetylation comprises acetylation of histone lysine residues H3K4, H3K9,H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof. In someembodiments, the HAT activator increases p53 acetylation. In someembodiments, the HDAC inhibitor increases p53 acetylation. In someembodiments, the HAT activator increases Bcl6 acetylation. In someembodiments, the HDAC inhibitor increases Bcl6 acetylation.

In some embodiments, treatment with a combination of a HAT activator anda HDAC inhibitor can result in a synergistic effect and a reduction incell viability of cancer cells. The use of a HAT activator incombination with a HDAC inhibitor can allow for a therapeuticallyeffective dose of either the HAT activator, or the HDAC inhibitor, orboth, to be administered at a lower dose.

Accordingly, one aspect of the invention is directed to methods fortreating cancer in a subject comprising administering to the subject aHAT activator and a HDAC inhibitor.

In some embodiments, the HAT activator is

In some embodiments, the HDAC inhibitor is romidepsin, vorinostat,belinostat, panobinostat, entinostat, mocetinostat, abexinostat,quisinostat, or gavinostat. In another embodiment, the HDAC inhibitor ischidamide, resminostat, givinostat, or kevetrin.

Eukaryotic DNA is highly organized and packaged into the nucleus. Theorganization and packaging are achieved through the addition ofproteins, including core histones H2A, H2B, H3 and H4, which form acomplex structure, the chromatin, together with DNA (see, for example,WO 2011/072243 and references cited therein). The modification of corehistones is of fundamental importance to conformational changes of thechromatin. The level of acetylation is related to transcriptionactivity, and then the acetylation induces an open chromatinconfirmation that allows the transcription machinery access topromoters. Histone deacetylase (HDAC) and histone acetyltransferase(HAT) are enzymes that influence transcription by selectivelydeacetylating or acetylating the F-amino groups of lysine located nearthe amino termini of core histone proteins. Chromatin acetylationcorrelates with transcriptional activity (euchromatin), whereasdeacetylation correlates with gene silencing. Further details on HAT,HDAC, chromatin, HAT activators and their role in neurodegenerativediseases and cancer can be found in WO 2011/072243; WO 2012/088420; andU.S. Patent Publication No. 2013/0121919, each incorporated by referenceherein in its entirety.

In some embodiments, the HAT modulator compound of the invention isdirected to GCN5, GCN5L, HAT1, PCAF, or a combination thereof. Examplesof HATs include, but are not limited to GCN5, GCN5L, PCAF, HAT1, ELP3,HPA2, ESA1, SAS2, SAS3, TIP60, HBO1, MOZ, MORF, MOF, SRC1, SRC3, TIF2,GRIP1, ATF-2 [see Lee and Workman (2007) Nat Rev Mol Cell Biol.,8(4):284-95, Marmorstein (2001) J Molec Biol. 311: 433-444; and Kimuraet al., (2005) J Biochem. 138(6): 647-662, which are each herebyincorporated by reference in their entireties]. In some embodiments, theHAT modulator comprises a protein that possesses intrinsic HAT activity,such as nuclear receptor co-activators (for example, CBP/p300 and Taf1).In some embodiments, the acetylation of H2, H3, and/or H4 histones isincreased. In some embodiments, the HAT modulator compound is a compoundof formula (I). In some embodiments, the HAT modulator compound isselected from the group consisting of:

Modulation of the acetylation state of histones, transcription factors,and other regulatory proteins is known to influence their activitywithin cancer and inflammatory cells. The acetylation state of a proteinis controlled by the activity of two main groups of enzymes, histonedeacetylases (HDAC) and histone acetyl transferases (HAT). The HDACremoves acetyl-groups while the HATs transfer acetyl-groups to theprotein of interest. Classically, modulation of acetylation status isknown to influence the condensation of chromatin. In cancer, histonesare deacetylated maintaining a condensed chromatin structure, and atranscriptionally silenced state. This transcriptional inactivation ismediated by HDACs which remove acetyl groups from histone tails,maintain a condensed chromatic structure. Inhibitors of HDACs helpmaintain transcriptionally active chromatin, theoretically allowing forexpression of tumor suppressor genes. Two HDAC inhibitors have beenapproved for the treatment of cancer, including vorinostat andromidepsin, currently FDA approved for the treatment of cutaneous T-celllymphoma and peripheral T-cell lymphoma.

Since these approvals, the pharmacology of this class of drugs has beenextensively studied. One observation that has evolved is that histonesare not the only targets of acetylation. It is now accepted thatpost-translational acetylation of intracellular proteins such as tumorsuppressors (p53) and oncogenes (Bcl6) plays a critical role ininfluencing their activity. It has been established that there is anetwork of proteins and enzymes that can be modified by acetylation, nowcollectively referred to as the acetylome. It has been shown thatmodulation of key intracellular proteins with HDAC inhibitors can leadto profound effects in lymphoma cell lines, in mouse models of lymphomaand in patients with drug-resistant lymphoma. Treatment with HDACinhibitors, like vorinostat, can inactivate the oncogene, Bcl6, whilesimultaneously activating the tumor suppressor, p53. The tumor suppressp53 plays an important role in many cancers, and mutations in p53 arecritical in the development of many cancers. Enhancement of p53 activitythrough acetylation protects the tumor suppressor from proteosomaldegradation and stimulates induction of apoptosis.

It has also recently been recognized that many patients with diffuselarge B-cell lymphoma and follicular lymphoma, the two most commonsubtypes of lymphoma, harbor inactivating mutations in one of twofamilies of HAT enzymes, CREBBP and p300. These mutations portend a moreaggressive phenotype of disease, and shortened survival in mouse models.These mutations are mostly heterozygous, suggesting that the normalhaploallele may still be amenable to modification, potentially reversingthis malignant phenotype. These mutations have also been identified inB-cell derived acute leukemias.

Given the clinical success of HDAC inhibitors, and the specific HATmutations in lymphomas, HAT activators may modify the acetylation stateof the proteome and may therefore represent a rational therapeutictarget for cancer. Furthermore, combined targeting of acetylationthrough HAT activation and HDAC inhibition may induce profoundpost-translational modification of key regulatory proteins and‘acetylation stress,’ leading to the induction of programmed cell death.

Relapsed and refractory T-cell lymphoma continues to be a rare butextraordinarily aggressive disease. HDAC inhibitors are approved for usein peripheral T-cell lymphoma and cutaneous T-cell lymphoma. Inaddition, diffuse large B-cell lymphoma and follicular lymphomas are thetwo most common subtypes of lymphoma and harbor heterogenousinactivating mutations in HATs. Without being bound by theory, theeffects of these mutations can be mitigated by enhancement of theireffect through pharmacologic modification. Treatment with HAT activatorsin cells with HAT mutations can reverse the malignant phenotype. Thesemutations can not be modulated by existing HDAC inhibitors.

Histone deacetylase inhibitors have activity on a set of enzymes thatremove acetyl groups from histones and transcription factors. AlthoughHAT activators and HDAC inhibitors have converse mechanisms of action,their end result is to enforce acetylation of histones and transcriptionfactors. HAT activators have a converse mechanism of action from knownagents with similar effects. They can reverse the malignant genotype ofmutated HATs in follicular and diffuse large B-cell lymphomas as well asB-cell derived acute leukemias. These actions would not be possible withHDAC inhibitors alone. HAT activators can be used to overcomeinactivating mutations of HATs, which is not possible with any known orexisting technology. In some embodiments, a HAT modulator can enhanceHDAC inhibitor activity. They may reverse a malignant phenotype ofdiffuse large 3-cell lymphoma and follicular lymphoma harboring FIATmutations. HAT activators can activate key tumor suppressor proteinssuch as p53.

In some embodiments, the invention described herein can be used to treatinflammatory diseases such as lupus, rheumatoid arthritis, and Sjogren'ssyndrome, as well as, a host of neurodegenerative diseases includingAlzheimer's Disease, Huntington disease, Friederich Ataxia, and others.

In some embodiments, there is also provided a method for reducing theproliferation of a cancer cell or cells comprising contacting thecell(s) with a HAT modulator and a HDAC modulator. In similarembodiments there is provided a use of a HAT modulator and a HDACmodulator for reducing the proliferation of a cancer cell or cells. Insome embodiments, there is provided a method for inducing cell death ina cancer cell or cells comprising contacting the cell(s) with a HATmodulator and a HDAC modulator. In similar embodiments there is provideda use of a HAT modulator and a HDAC modulator for inducing cell death ina cancer cell or cells. In some embodiments, the HAT modulator is a HATactivator. In some embodiments, the HAT modulator is a HAT inhibitor. Inother embodiments, the HDAC modulator is a HDAC inhibitor. In otherembodiments, the HDAC inhibitor is a HDAC activator. In someembodiments, the invention is directed to the combination of one or moreHAT activators and one or more HDAC inhibitors. In some embodiments, theinvention is directed to the combination of a HAT activator and a HDACinhibitor. In some embodiments, the cancer cell may be in vivo or invitro. In some embodiments, the cancer cell may be a precancerous cell.In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin'slymphoma, B cell lymphoma, T cell lymphoma, or follicular lymphoma. Inother embodiments, the B cell lymphoma is diffuse large B-cell lymphoma.In further embodiments, the diffuse large B-cell lymphoma is a germinalcenter-derived diffuse large B cell lymphoma, an activatedB-cell-derived (ABC) diffuse large B-cell lymphoma, or a non-germinalcenter diffuse large B cell lymphoma. In some embodiments, the HDACinhibitor is romidepsin, vorinostat, belinostat, panobinostat,entinostat, mocetinostat, abexinostat, quisinostat or gavinostat.

Increasing histone acetylation has been shown to improve outcome in awide variety of diseases as diverse as asthma, infectious disease andpsychiatric diseases. Although clinical trials of several HDACinhibitors are currently underway, the alternative strategy where byhistone acetylation is increased by HAT modulator has not beenextensively explored. For example, compounds in U.S. Patent PublicationNo. US2009/076155 and PCT Publication No. WO2004/053140 (which are eachhereby incorporated by reference in their entireties) have poorsolubility and membrane permeability. Other HAT modulators are describedin WO 2011/072243; WO 2012/088420; and U.S. Patent Publication No.2013/0121919, each incorporated by reference herein in its entirety.

Several HDACi are in trials for cancer some of which are, for example,4SC-202 (Nycomed, Germany), which is in a Preclinical stage; AR-42 (Arnotherapeutics, Parsippany, N.J.) which is in a Preclinical stage;Belinostat (TopoTarget, Rockaway, N.J.) which is in Phase II clinicaltrials; and Entinostat (Bayer Schering) which is in Phase II clinicaltrials. For example, in Table 3 of Lane and Chabner (2009, J ClinOncol., 27(32):5459-68; incorporated by reference in its entirety),selected clinical trials of HDAC inhibitors are discussed, which includeVorinostat, Depsipeptide, and MGCD0103. In Table 2 of Lane and Chabner(2009, J Clin Oncol., 27(32):5459-68; incorporated by reference in itsentirety), selected HDAC inhibitors in clinical use or development arediscussed, which include hydroxamic acid compounds (e.g., Vorinostat,Trichostatin A, LAQ824, Panobinostat, Belinostat, and ITF2357), cyclictetrapeptide compounds (e.g., Depsipeptide), benzamide compounds (e.g.,Entinostat and MGCD0103), and short-chain aliphatic acid compounds(e.g., valproic acid, phenyl butyrate, and pivanex). Other HDACinhibitors include, but are not limited to, romidepsin, vorinostat,belinostat, panobinostat, entinostat, mocetinostat, abexinostat,quisinostat or gavinostat. In another embodiment, the HDAC inhibitor ischidamide, resminostat, givinostat, or kevetrin.

Vorinostat, also known as Zolinza® (Merck, Whitehouse Station, N.J.)inhibits the enzymatic activity of histone deacetylases HDAC1, HDAC2 andHDAC3 (Class I) and HDAC6 (Class II). In vitro, vorinostat causes theaccumulation of acetylated histones and induces cell cycle arrest and/orapoptosis of some transformed cells. Vorinostat is indicated for thetreatment of cutaneous manifestations in patients with cutaneous T-celllymphoma (CTCL) who have persistent or recurrent disease on or followingtwo systemic therapies. Further details on vorinostat are discussed inthe Zolina® label, incorporated by reference in its entirety.

Romidepsin, also known as Istodax® (Celgene, Summit, N.J.) is a bicyclicdepsipeptide and inhibits histone deacetylases. In vitro, romidepsincauses the accumulation of acetylated histones and induces cell cyclearrest and/or apoptosis of some cancer cell lines. Romidepsin isindicated for the treatment of cutaneous T-cell lymphoma (CTCL) inpatients who have received at least one prior systemic therapy and forthe treatment of peripheral T-cell lymphoma (PTCL) in patients who havereceived at least one prior systemic therapy. Further details onromidepsin are discussed in the Istodax® label, incorporated byreference in its entirety.

Some HDACi are or were being developed for neurological diseases, suchas an HDACi from Merck (Whitehouse Station, N.J.) that is being used forthe treatment of neurodegenerative diseases; and HDACi from TopoTarget(Rockaway, N.J.) that was being used for the treatment of Huntington'sdisease, now discontinued; isovaleramide NPS-1776 (NPS Pharmaceutical,Bedminster, N.J.) that was being used for bipolar disorder, epilepsy,and migraines, now discontinued; and a histone acetyltransferaseinhibitor for cancer from TopoTarget A/S (København, Denmark), which wasdiscontinued in the preclinical stage.

In some embodiments, the HAT modulator and HDAC modulator combinationsmay have a synergistic effect, for example, HAT activator and HDACinhibitor combinations can result in increased histone acetylationcompared to histone acetylation of the HAT activator or HDAC inhibitoralone. Combined targeting of acetylation through HAT activation and HDACinhibition may induce profound post-translational modification of keyregulatory proteins and ‘acetylation stress,’ leading to the inductionof programmed cell death. In some embodiments, HAT activator and HDACinhibitor combinations may lead to acetylation of p53, acetylation ofBcl6 and/or induction of p21.

In some embodiments, the methods include administering a HAT activatorand HDAC inhibitor to a subject, wherein the subject is wildtype for allHAT enzymes. In some embodiments, the methods include administering aHAT activator and HDAC inhibitor to a subject, wherein the subject hasat least one mutant HAT enzyme gene. In some embodiments, the methodsinclude administering a HAT activator and HDAC inhibitor to a subject,the subject has a wildtype EP300 and wildtype CREBBP gene. In anotherembodiment, the subject has a wildtype EP300 and CREBBP mutant gene. Ina specific embodiment, the CREBBP mutant gene comprises a mismatchmutation. In another specific embodiment, the CREBBP mutant genecomprises a truncation mutation. In another specific embodiment, theCREBBP mutant gene comprises at least one point mutation. In a specificembodiment, the mutation is on only one allele of CREBBP.

In another embodiment, the subject has a mutant EP300 and CREBBPwildtype gene. In a specific embodiment, the EP300 mutant gene comprisesa mismatch mutation. In another specific embodiment, the EP300 mutantgene comprises a truncation mutation. In another specific embodiment,the EP300 mutant gene comprises at least one point mutation. In aspecific embodiment, the mutation is on only one allele of EP300.

In another embodiment, the subject has a mutant EP300 and CREBBP mutantgene. In a specific embodiment, the EP300 mutant gene and/or CREBBPmutant gene comprises a mismatch mutation. In another specificembodiment, the EP300 mutant gene and/or CREBBP mutant gene comprises atruncation mutation. In another specific embodiment, the EP300 mutantgene and/or CREBBP mutant gene comprises at least one point mutation. Ina specific embodiment, the EP300 mutant gene has a mismatch mutation andthe CREBBP mutant gene has a truncated mutation. In a specificembodiment, the mutation is on only one allele of CREBBP and/or on onlyone allele of EP300. In another embodiment, methods includeadministering a HAT activator and HDAC inhibitor to a subject for thetreatment and/or inhibition of lymphoma. In a specific embodiment, thelymphoma is diffuse large B-cell lymphoma and/or follicular lymphoma. Inanother embodiment, methods include administering a HAT activator andHDAC inhibitor to a subject for the treatment and/or inhibition ofB-cell derived acute leukemias. In a specific embodiment, the HATactivator

is

or a pharmaceutically acceptable salt thereof. In another specificembodiment, the HAT activator is

or a pharmaceutically acceptable salt thereof.

In another specific embodiment, the HDAC inhibitor is romidepsin.

In some embodiments, a HAT modulator compound can be used in combinationwith one or more HDAC modulators to treat a cancer in a subject in needthereof. In other embodiments, a HAT activator compound can be used incombination with one or more HDAC inhibitors to treat a cancer in asubject in need thereof. Non-limiting examples of cancers include B celllymphoma, colon cancer, lung cancer, renal cancer, bladder cancer, Tcell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, acute lymphocyticleukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lungcancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma,uterine cancer, renal cell carcinoma, hepatoma, adenocarcinoma, breastcancer, pancreatic cancer, liver cancer, prostate cancer, head and neckcarcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer,primary or metastatic melanoma, squamous cell carcinoma, basal cellcarcinoma, brain cancer, angiosarcoma, hemangiosarcoma, bone sarcoma,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, testicular cancer, uterinecancer, cervical cancer, gastrointestinal cancer, mesothelioma, Ewing'stumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, Waldenstroom'smacroglobulinemia, papillary adenocarcinomas, cystadenocarcinoma,bronchogenic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, lung carcinoma, epithelial carcinoma,cervical cancer, testicular tumor, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, retinoblastoma, leukemia,melanoma, neuroblastoma, small cell lung carcinoma, bladder carcinoma,lymphoma, multiple myeloma, follicular lymphoma and medullary carcinoma.

In some embodiments, the cancer is colon cancer, lung cancer, renalcancer, leukemia, CNS cancer, melanoma, ovarian cancer, breast cancer,or prostate cancer.

In some embodiments, the cancer is colon cancer, renal cancer, T cellleukemia, myeloma, leukemia, acute myeloid leukemia, acute lymphocyticleukemia, renal cell carcinoma, adenocarcinoma, glioblastoma, breastcarcinoma, prostate carcinoma, or lung carcinoma.

In some embodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin'slymphoma, B cell lymphoma, T cell lymphoma, or follicular lymphoma. Inother embodiments, the B cell lymphoma is diffuse large B-cell lymphoma.In further embodiments, the diffuse large B-cell lymphoma is a germinalcenter-derived diffuse large B cell lymphoma, an activatedB-cell-derived (ABC) diffuse large B-cell lymphoma, or a non-germinalcenter diffuse large B cell lymphoma.

In some embodiments, a HAT modulator compound can be used in combinationwith one or more HDAC modulators to treat a neurodegenerative disease ina subject in need thereof. In other embodiments, a HAT activatorcompound can be used in combination with one or more HDAC inhibitors totreat a neurodegenerative disease in a subject in need Non-limitingexamples of neurodegenerative diseases include Adrenoleukodystrophy(ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer'sdisease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia,Frontotemporal lobar degeneration, Huntington's disease, HIV-associateddementia, Kennedy's disease, Krabbe's disease, Lewy body dementia,Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, NiemannPick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick'sdisease, Primary lateral sclerosis, Prion diseases ProgressiveSupranuclear Palsy, Refsum's disease, Rett's syndrome, Tau-positiveFrontoTemporal dementia, Tau-negative FrontoTemporal dementia, Sandhoffdisease, Schilder's disease, Subacute combined degeneration of spinalcord secondary to Pernicious Anaemia, Spielmeyer-Vogt-Sjogren-Battendisease (also known as Batten disease), Spinocerebellar ataxia (multipletypes with varying characteristics), Spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, and Toxicencephalopathy.

In some embodiments, the neurodegenerative disease is selected fromAlzheimer's Disease, ALS, Parkinson's Disease, and Huntington's Disease.In some embodiments, the neurodegenerative disease is Alzheimer'sDisease. In some embodiments, the neurodegenerative disease isHuntington's Disease.

Epigenetic modifications including acetylation of histones maycontribute to gene expression changes important to learning and memory(Science 2010: 328(5979), 701-702; herein incorporated by reference inits entirety). Addition of acetyl groups to histones by histoneacyltransferases (HAT) enhances gene expression, while their removal byhistone deacetylases (HDAC) reduces gene expression. Reduction inhistone acetylation has recently been linked to age-induced memoryimpairment and various neurodegenerative diseases (Science 2010:328(5979), 701-702; herein incorporated by reference in its entirety).HDAC inhibitors have been shown to enhance memory in mice (Nature 459,55-60 (7 May 2009); herein incorporated by reference in its entirety).Although clinical trials of several HDAC inhibitors are currentlyunderway to try to prevent deacetylation, the alternative strategy ofincreasing histone acetylation by activating HAT has not beensignificantly explored. Histone acetylation is discussed in, forexample, U.S. Patent Publication Nos. 2010/0166781; 2010/0144885;2009/0076155; Neuroscience 2011, 194, 272-281; and J. Phys. Chem B 2007,111(17), 4527-4534 (each of which herein incorporated by reference inits entirety). Further details on neurodegenerative diseases, includingAlzheimer's disease, can be found in WO 2011/072243 and WO 2012/088420,each incorporated by reference herein in its entirety.

In some embodiments, the invention provides for compounds with histoneacetyltransferase activity which can be used in combination with one ormore HDAC modulators to treat patients with cancers or neurodegenerativediseases. In some embodiments, the compounds are HAT activators. In someembodiments, the compounds are HAT inhibitors. In some embodiments, theHDAC modulator is a HDAC activator. In some embodiments, the HDACmodulator is a HDAC inhibitor. In some embodiments, the compounds havegood HAT activation potency, high selectivity, reasonablepharmacokinetics and/or good permeability across the blood-brain-barrier(BBB). In some embodiments, these compounds can be used as therapy withdecreased side effects for AD patients. In some embodiments, thecompounds improve cognition or memory in AD and Alzheimer's-likepathologies, as well as minimize the side effects for subjects afflictedwith other neurodegenerative diseases. In some embodiments, thecompounds of the invention can also be developed as anti-cancertherapies. In some embodiments, acetylation of histone proteinsincreases gene expression in a subject resulting in enhanced memory andcognition.

In some embodiments, the invention provides a method for reducingamyloid beta (A) protein deposits in a subject in need thereof, themethod comprising administering to the subject a HAT activator and aHDAC inhibitor. In some embodiments, the subject exhibits abnormallyelevated levels of amyloid beta plaques. In some embodiments, thesubject is afflicted with Alzheimer's disease, Lewy body dementia,inclusion body myositis, or cerebral amyloid angiopathy.

In further embodiments, the invention provides for the utilization ofHAT agonists in combination with one or more HDAC modulators as memoryenhancers in normal subjects (for example, a subject not afflicted witha neurodegenerative disease). In further embodiments, the inventionprovides for the utilization of HAT agonists in combination with one ormore HDAC modulators as memory enhancers in aging subjects (for example,a subject who is >55 years old). In further embodiments, the inventionprovides for the utilization of HAT agonists in combination with one ormore HDAC modulators as memory enhancers for other conditions associatedwith cognitive decrease/impairment. In some embodiments, the HDACmodulator is a HDAC activator. In some embodiments, the HDAC modulatoris a HDAC inhibitor. Non-limiting examples of conditions associated withcognitive decrease/impairment include a variety of syndromes associatedwith mental retardation and syndromes associated with learningdisabilities, Parkinson's disease, Pick's disease, a Lewy body disease,amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakobdisease, Down syndrome, multiple system atrophy, neuronal degenerationwith brain iron accumulation type I (Hallervorden-Spatz disease), pureautonomic failure, REM sleep behavior disorder, mild cognitiveimpairment (MCI), cerebral amyloid angiopathy (CAA), mild cognitivedeficits, aging, vascular dementias mixed with Alzheimer's disease, aneurodegenerative disease characterized by abnormal amyloid deposition,and any combination thereof.

In some embodiments, the invention provides methods for identifying acombination of one or more HAT modulators and one or more HDACmodulators that can acetylate histone proteins thus increasing geneexpression in a subject resulting in enhanced memory and cognition. Insome embodiments, the invention provides methods for identifying acombination of one or more HAT activators and one or more HDACinhibitors can acetylate histone proteins thus increasing geneexpression in a subject resulting in enhanced memory and cognition.

To shrink the candidate pool of HAT modulator and HDAC modulatorcombinations to be tested in animal models of neurodegenerativediseases, such as animals that exhibit elevated levels of inclusionbodies, for example Aβ accumulation animal models (e.g., animal modelsof AD), or, for example, a mouse model for Huntington's disease, HATmodulators or HDAC modulators can first be screened or selected based ontheir possession of certain characteristics, such as having one or moreof: an EC₅₀ no greater than about 100 nM; a histone acetylation activityin vitro; and the ability to penetrate the BBB. HAT modulator and HDACmodulator combinations can first be screened or selected based on theirpossession of certain characteristics, such as having a histoneacetylation activity in vitro or resulting in increased histoneacetylation in vitro compared to histone acetylation in vitro of the HATmodulator or HDAC modulator alone.

In some embodiments, the candidate pool of HAT modulator and HDACmodulator combinations can be tested in animal models ofneurodegenerative diseases, such as, but not limited to, animals thatexhibit elevated levels of inclusion bodies, for example A accumulationanimal models (e.g., animal models of AD), or a mouse model forHuntington's disease to determine whether they increase gene expressionin a subject resulting in enhanced memory and cognition. As used herein,a HAT activator compound does not necessarily preclude the possibilitythat the compound may also be able to inhibit other HATs. As usedherein, a HDAC inhibitor compound does not necessarily preclude thepossibility that the compound may also be able to activate other HATs.

In some embodiments, the compounds of the invention are HAT modulators.The term “modulate”, as it appears herein, refers to a change in theactivity or expression of a protein molecule. For example, modulationcan cause an increase or a decrease in protein activity, bindingcharacteristics, or any other biological, functional, or immunologicalproperties of a secretase protein molecule. In some embodiments, thecompounds activate HAT. In some embodiments, the compounds inhibit HAT.

In some embodiments, the compounds of the invention are HDAC modulators.The term “modulate”, as it appears herein, refers to a change in theactivity or expression of a protein molecule. For example, modulationcan cause an increase or a decrease in protein activity, bindingcharacteristics, or any other biological, functional, or immunologicalproperties of a secretase protein molecule. In some embodiments, thecompounds inhibit HDAC. In some embodiments, the compounds activateHDAC.

A HAT modulator compound can be a compound that increases the activityand/or expression of a HAT molecule (e.g., GCN5, GCN5L, PCAF, or HAT1)in vivo and/or in vitro. HAT modulator compounds can be compounds thatexert their effect on the activity of a HAT protein via the expression,via post-translational modifications, or by other means. In someembodiments, a HAT modulator compound increases HAT protein or mRNAexpression, or acetyltransferase activity by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, at least about 99%, or 100%.

A HDAC modulator compound can be a compound that decreases the activityand/or expression of a HDAC molecule in vivo and/or in vitro. HDACmodulator compounds can be compounds that exert their effect on theactivity of a HDAC protein via the expression, via post-translationalmodifications, or by other means. In some embodiments, a HDAC modulatorcompound decreases HDAC protein or mRNA expression, ordeacetyltransferase activity by at least about 10%, at least about 20%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 97%, at least about 99%, or 100%.

Test compounds or agents that bind to a HAT molecule (such as GCN5,GCN5L, PCAF, or HAT1), and/or have a stimulatory effect on the activityor the expression of a HAT molecule, can be identified by variousassays. The assay can be a binding assay comprising direct or indirectmeasurement of the binding of a test compound or a known HAT ligand tothe active site of a HAT protein. The assay can also be an activityassay comprising direct or indirect measurement of the activity of a HATmolecule. The assay can also be an expression assay comprising direct orindirect measurement of the expression of a HAT mRNA or protein. Thevarious screening assays can be combined with an in vivo assaycomprising measuring the effect of the test compound on cognitive andsynaptic function in an animal model for neurodegenerative disorders,such as, but not limited to, AD or Huntington's Disease. The assay canbe an assay comprising measuring the effect of the test compounds oncell viability. In one embodiment, the cells are cancer cells, such as,but not limited to B-cell lymphoma cell lines, or T-cell lymphoma celllines (e.g. Ly1, Ly7, Ly10, SU-DHL2, HH, or H9 cell lines).

The inhibitors of the expression of a HAT molecule can be identified viacontacting a HAT-positive cell or tissue with a test compound anddetermining the expression of a HAT protein or HAT mRNA in the cell. Theprotein or mRNA expression level of a HAT molecule in the presence ofthe test compound can be compared to the protein or mRNA expressionlevel of a HAT protein in the absence of the test compound. The testcompound can then be identified as an inhibitor of expression of a HATprotein (such as GCN5, GCN5L, PCAF, or HAT1) based on this comparison.In other words, the test compound can also be a HAT inhibitor compound(such as an antagonist).

Activators of the expression of a HAT molecule can also be identifiedvia contacting a HAT-positive cell or tissue with a test compound anddetermining the expression of a HAT protein or HAT mRNA in the cell. Theprotein or mRNA expression level of a HAT molecule in the presence ofthe test compound can be compared to the protein or mRNA expressionlevel of a HAT protein in the absence of the test compound. The testcompound can then be identified as an activator of expression of a HATprotein (such as GCN5, GCN5L, PCAF, or HAT1) based on this comparison.For example, when expression of HAT protein or mRNA is statistically orsignificantly more in the presence of the test compound than in itsabsence, the compound is identified as an activator of the expression ofa HAT protein or mRNA. In other words, the test compound can also be aHAT activator compound (such as an agonist). The expression level of aHAT protein or mRNA in cells can be determined by methods describedherein.

Determining the ability of a test compound to bind to a HAT molecule, aHDAC molecule or a variant thereof can be accomplished using real-timeBimolecular Interaction Analysis (BIA) [McConnell, (1992); Sjolander,S., and Urbaniczky, C. Integrated fluid handling system for biomolecularinteraction analysis. Anal. Chem. 1991, 63, 2338-2345; hereinincorporated by reference in its entirety]. BIA is a technology forstudying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIA-Core™). Changes in optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In some embodiments, the invention provides for compounds that bind to aHAT activator protein, such as GCN5, GCN5L, PCAF, or HAT1. Thesecompounds can be identified by the screening methods and assaysdescribed herein, and enhance the activity or expression of HATactivator proteins.

Test compounds or agents that bind to a HAT molecule and/or have astimulatory effect on the activity or the expression of a HAT molecule,can be combined with one or more test compounds or agents that bind to aHDAC molecule. The assay can be an activity assay comprising direct orindirect measurement of the activity of a HAT molecule and/or a HDACmolecule. The assay can also be an expression assay comprising direct orindirect measurement of the expression of a HAT mRNA or protein and/or aHDAC mRNA or protein. The various screening assays can be combined withan in vivo assay comprising measuring the effect of a HAT activator anda HDAC inhibitor on cognitive and synaptic function in an animal modelfor neurodegenerative disorders, such as, but not limited to, AD orHuntington's Disease. The assay can be an assay comprising measuring theeffect of the test compounds on cell viability. In one embodiment, thecells are cancer cells, such as, but not limited to B-cell lymphoma celllines, or T-cell lymphoma cell lines. In one embodiment, the effect of aHAT activator and one or more HDAC inhibitors in combination is comparedto the effect of a HAT activator or HDAC inhibitor alone.

Synthesis of representative HAT activators is disclosed, for example, inWO 2011/072243; WO12/088420 and US Patent Pub. No. 2013/0121919; eachherein incorporated by reference in its entirety.

In some embodiments, the HAT activator is a compound of formula (I),

wherein,Ar is

R^(a) is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;R^(b) is H, OH, halogen, C₁-C₆-alkyl, —(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl),C₁-C₆-haloalkyl, C₂-C₆-alkenyl, C₃-C₈-cycloalkyl, C₂-C₆-heteroalkyl,C₃-C₈-heterocycloalkyl, aryl, heteroaryl, O—(C₁-C₆-alkyl),O—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), O—(C₁-C₆-haloalkyl),O—(C₃-C₈-cycloalkyl), O—(C₂-C₆-alkenyl), O—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₁-C₆-alkyl), N(R¹⁰)—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl),N(R¹⁰)—(C₃-C₈-cycloalkyl), SH, S—(C₁-C₆-alkyl),S—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), SO₂—(C₁-C₆-alkyl),SO₂—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)-N(R¹⁰)₂,O—(C₂-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻, O—(C₃-C₈-cycloalkyl)-N(R¹⁰)₂,N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-R³,O—(C₁-C₆-alkyl)-R³, O—(C₃-C₈-cycloalkyl)-R³, N(R¹⁰)—(C₁-C₆-alkyl)-R³,O-aryl, or O-heteroaryl;R^(c) is H, —(C₁-C₆-alkyl), O—(C₁-C₆-alkyl), C(═O)NH-phenyl, whereinphenyl is substituted with one or more halo or haloalkyl;R^(d) is H, OH, halogen, C₁-C₁₆-alkyl, C₁-C₁₆-haloalkyl,O—(C₃-C₈-cycloalkyl), O—(C₃-C₈-heterocycloalkyl), O—(C₂-C₆-alkenyl),O—(C₁-C₆-alkyl), O—(C₁-C₆-alkyl)-phenyl, O—(C₂-C₆-alkyl)-N(R¹⁰)₂,O—(C₂-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻, —(C₁-C₆-alkyl)-R³,O—(C₁-C₆-alkyl)-R³, OS(C₁-C₆-alkyl), N(R¹⁰)—(C₁-C₆-alkyl)-R³, —N(R¹⁰)—(C₁-C₆-alkyl),—N(R¹⁰)—(C₂-C₆-alkenyl), —N(R¹⁰)—(C₃-C₈-cycloalkyl),—N(R¹⁰)—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂—(C₁-C₆-alkyl)-N(R¹⁰)₂,S—(C₂-C₆-alkyl)-N(R¹⁰)₂, OCH₂C(O)O(C₁-C₆-alkyl), O-aryl, N-aryl,O-heteroaryl, or N-heteroaryl;U¹-U⁴ are independently N or CR^(a), wherein U¹-U⁴ are not each N;V is a bond, N or CR^(c);W and Z are independently N or CR¹;X is —CO—, —CON(R¹⁰)—, —CON(R¹⁰)(CH₂)_(n)—, —(CH₂)_(n)CON(R¹⁰)—,—(CH₂)_(n)CON(R¹⁰)(CH₂)_(n)—, —SON(R¹⁰)—, —SON(R¹⁰)(CH₂)_(n)—,—SO₂N(R¹⁰)—, —SO₂N(R¹⁰)(CH₂)_(n)—, —N(R¹⁰)C(═O)N(R¹⁰)—, —N(R¹⁰)CO—,—N(R¹⁰)CO(CH₂)_(n)—, or —N(R¹⁰)CO(CH₂)_(n)—, —(CH₂)_(n)N(R¹⁰)—, —C═N—;orAr and X together form

Y is a bond, N or CR²;R¹ is H, halogen, O—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)N(R¹⁰)₂;R² is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;R³ is cycloalkylamino, optionally containing a heteroatom selected fromN(R¹⁰), O and S;R¹⁰ is independently H, —(C₁-C₄-alkyl), —(C₁-C₄-haloalkyl),—(C₃-C₈-cycloalkyl), —(C₃-C₈-heterocycloalkyl), aryl or heteroaryl;

-   -   is a double bond and R¹¹ is O, or    -   is a single bond and R¹¹ is —(C₁-C₆-alkyl),        —(C₁-C₆-alkyl)-N(R¹⁰)₂, or —(C₁-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻; and        each n is independently an integer from 1-4, or a        pharmaceutically acceptable salt thereof.

In some embodiments, the HAT activator is a compound of formula (I),

wherein,Ar is

R^(a) is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;R^(b) is H, OH, C₁-C₆-alkyl, —(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl),C₁-C₆-haloalkyl, C₂-C₆-alkenyl, C₃-C₈-cycloalkyl, C₂-C₆-heteroalkyl,C₃-C₈-heterocycloalkyl, aryl, heteroaryl, O—(C₁-C₆-alkyl),O—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), O—(C₁-C₆-haloalkyl),O—(C₃-C₈-cycloalkyl), O—(C₂-C₆-alkenyl), O—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₁-C₆-alkyl), N(R¹⁰)—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl),N(R¹⁰)—(C₃-C₈-cycloalkyl), SH, S—(C₁-C₆-alkyl),S—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), SO₂—(C₁-C₆-alkyl),SO₂—(C₁-C₆-alkyl)CO₂—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)-N(R¹⁰)₂,O—(C₂-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻, O—(C₃-C₈-cycloalkyl)-N(R¹⁰)₂,N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-R³,O—(C₁-C₆-alkyl)-R³, O—(C₃-C₈-cycloalkyl)-R³, N(R¹⁰)—(C₁-C₆-alkyl)-R³,O-aryl, or O-heteroaryl;R^(c) is H, —(C₁-C₆-alkyl), O—(C₁-C₆-alkyl), C(═O)NH-phenyl, whereinphenyl is substituted with one or more halo or haloalkyl;R^(d) is H, OH, C₁-C₁₆-alkyl, C₁-C₁₆-haloalkyl, O—(C₃-C₈-cycloalkyl),O—(C₃-C₈-heterocycloalkyl), O—(C₂-C₆-alkenyl), O—(C₁-C₆-alkyl),O—(C₁-C₆-alkyl)-phenyl, O—(C₂-C₆-alkyl)-N(R¹⁰)₂, O—(C₂-C₆-alkyl)-N(R¹⁰)₃⁺halogen⁻, —(C₁-C₆-alkyl)-R³, O—(C₁-C₆-alkyl)-R³, OS(C₁-C₆-alkyl), N(R¹⁰)—(C₁-C₆-alkyl)-R³, —N(R¹⁰)—(C₁-C₆-alkyl),—N(R¹⁰)—(C₂-C₆-alkenyl), —N(R¹⁰)—(C₃-C₈-cycloalkyl),—N(R¹⁰)—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂—(C₁-C₆-alkyl)-N(R¹⁰)₂,S—(C₂-C₆-alkyl)-N(R¹⁰)₂, OCH₂C(O)O(C₁-C₆-alkyl), O-aryl, N-aryl,O-heteroaryl, or N-heteroaryl;U¹-U⁴ are independently N or CR^(a), wherein U¹-U⁴ are not each N;V is a bond, N or CR^(c);W and Z are independently N or CR¹;X is —CO—, —CON(R¹⁰)—, —CON(R¹⁰)(CH₂)_(n)—, —(CH₂)_(n)CON(R¹⁰)—,—(CH₂)_(n)CON(R¹⁰)(CH₂)_(n)—, —SON(R¹⁰)—, —SON(R¹⁰)(CH₂)_(n)—,—SO₂N(R¹⁰)—, —SO₂N(R¹⁰)(CH₂)_(n)—, —N(R¹⁰)C(═O)N(R¹⁰)—, —N(R¹⁰)CO—,—N(R¹⁰)CO(CH₂)_(n)—, or —N(R¹⁰)CO(CH₂)_(n)—, —(CH₂)_(n)N(R¹⁰)—, —C═N—;orAr and X together form

Y is a bond, N or CR²;R¹ is H, halogen, O—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)N(R¹⁰)₂;R² is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;R³ is cycloalkylamino, optionally containing a heteroatom selected fromN(R¹⁰), O and S;R¹⁰ is independently H, —(C₁-C₄-alkyl), —(C₁-C₄-haloalkyl),—(C₃-C₈-cycloalkyl), —(C₃-C₈-heterocycloalkyl), aryl or heteroaryl;

-   -   is a double bond and R¹¹ is O, or    -   is a single bond and R¹¹ is —(C₁-C₆-alkyl),        —(C₁-C₆-alkyl)-N(R¹⁰)₂, or —(C₁-C₆-alkyl)-N(R¹⁰)₃ ⁺halogen⁻; and        each n is independently an integer from 1-4, or a        pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is a compound offormula (Ia),

wherein,Ar is

R^(a) is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;R^(b) is H, C₁-C₆-alkyl, C₂-C₆-alkenyl, C₃-C₈-cycloalkyl,C₂-C₆-heteroalkyl, C₃-C₈-heterocycloalkyl, aryl, heteroaryl,O—(C₁-C₆-alkyl), O—(C₃-C₈-cycloalkyl), O—(C₂-C₆-alkenyl),O—(C₃-C₈-heterocycloalkyl), N(R¹⁰)—(C₁-C₆-alkyl),N(R¹⁰)—(C₃-C₈-cycloalkyl), S—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)-N(R¹⁰)₂,O—(C₃-C₈-cycloalkyl)-N(R¹⁰)₂, N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂,—(C₁-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-R³, O—(C₁-C₆-alkyl)-R³,O—(C₃-C₈-cycloalkyl)-R³, N(R¹⁰)—(C₁-C₆-alkyl)-R³, O-aryl, orO-heteroaryl;R^(c) is H, —(C₁-C₆-alkyl), O—(C₁-C₆-alkyl), C(═O)NH-phenyl, whereinphenyl is substituted with one or more halo or haloalkyl;R^(d) is H, OH, —(C₁-C₆-alkyl), O—(C₃-C₈-cycloalkyl),O—(C₃-C₈-heterocycloalkyl), O—(C₂-C₆-alkenyl), O—(C₁-C₆-alkyl),O—(C₂-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-R³, O—(C₁-C₆-alkyl)-R³,N(R¹⁰)—(C₁-C₆-alkyl)-R³, —N(R¹⁰)—(C₁-C₆-alkyl), —N(R¹⁰)—(C₂-C₆-alkenyl),—N(R¹⁰)—(C₃-C₈-cycloalkyl), —N(R¹⁰)—(C₃-C₈-heterocycloalkyl),N(R¹⁰)—(C₂-C₆-alkyl)-N(R¹⁰)₂, —(C₁-C₆-alkyl)-N(R¹⁰)₂,S—(C₂-C₆-alkyl)-N(R¹⁰)₂, OCH₂C(O)O-alkyl, O-aryl, N-aryl, O-heteroaryl,or N-heteroaryl;W and Z are independently N or CR¹;X is —CO—, —CON(R¹⁰)—, —CON(R¹⁰)(CH₂)_(n)—, —(CH₂)_(n)CON(R¹⁰)—,—(CH₂)_(n)CON(R¹⁰)(CH₂)_(n)—, —SON(R¹⁰)—, —SON(R¹⁰)(CH₂)_(n)—,—SO₂N(R¹⁰)—, —SO₂N(R¹⁰)(CH₂)_(n)—, —N(R¹⁰)C(═O)N(R¹⁰)—, —N(R¹⁰)CO—,—N(R¹⁰)CO(CH₂)_(n)—, or —N(R¹⁰)CO(CH₂)_(n)—, —(CH₂)_(n)N(R¹⁰)—, —C═N—;orAr and X together form

Y is N or CR²;R¹ is H, halogen, O—(C₁-C₆-alkyl), O—(C₂-C₆-alkyl)N(R¹⁰)₂;R² is H, C₁-C₆-alkyl, C₁-C₆-haloalkyl, O—(C₁-C₆-alkyl),O—(C₁-C₆-haloalkyl), halogen, CN, or NO₂;R³ is cycloalkylamino, optionally containing a heteroatom selected fromN(R¹⁰), O and S;R¹⁰ is independently H, —(C₁-C₄-alkyl), —(C₁-C₄-haloalkyl),—(C₃-C₈-cycloalkyl), —(C₃-C₅-heterocycloalkyl), aryl or heteroaryl; andeach n is independently an integer from 1-3.

In some embodiments, the compound of formula (I) is

wherein R is H, Methyl, Ethyl, n-Propyl, Isopropyl, n-butyl, t-butyl,C₈H₁₈, C₁₅H₂₆, C₁₅H₂₈, C₁₅H₃₀, or C₁₅H₃₂.

In some embodiments, the compound of formula (I) is

In some embodiments, the compound of formula (I) is

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is

or a pharmaceutically acceptable salt thereof.

Exemplary methods of preparation of compounds of formula (I) are shownin Scheme A.

Compound A can be treated with a primary amine in the presence of atertiary amine in a solvent such as toluene and heated to obtaincompounds B. Compounds B can be alkylated using an alkyl halide oraminoalkyl halide in the presence of a base such as potassium carbonatein a solvent such as N,N-dimethylformamide to obtain compounds C. In thecase of compounds C that contain amino-alkyl groups, treatment withmethyl iodide can be performed to obtain the methylamino salt.

Further exemplary methods of preparation of compounds of formula (I) areshown in Scheme B.

Compounds D can be treated with an amino-aldehyde in the presence of anacid such as phosphoric acid in a solvent such as toluene to obtaincompounds E. Alternatively, compounds D can be treated with a formatesource such as isopropyl chloroformate or triphosgene in a solvent suchas pyridine to obtain compounds F.

Pharmaceutically acceptable salts are known in the art, and can beselected from those listed in Berge, et al. [“Pharmaceutical Salts,” J.Pharm. Sci., 66(1):1-19 (January 1977); herein incorporated by referencein its entirety]. In some embodiments, a pharmaceutically acceptablesalt of a compound of formula (I) is an acid addition salt, for examplea hydrohalide (such as hydrochloride or hydrobromide), sulfate, orphosphate salt. In some embodiments, a pharmaceutically acceptable saltof a compound of formula (I) is a base addition salt, for example asodium, potassium, calcium, or ammonium salt. In some embodiments, thebase addition salt is a tetrafluoroboro salt.

In some embodiments, the invention provides methods for reducinginclusion bodies (e.g., amyloid beta (Aβ) protein deposits, native andphosphorylated Tau proteins, native and phosphorylated alpha-synuclein,lipofuscin, cleaved TARDBP (TDB-43), or a combination thereof) in asubject afflicted with a neurodegenerative disease (e.g., a AD,Huntington's Disease, or Parkinson's Disease) by administering any oneof the HAT modulator compounds having formula (I) and a HDAC modulator.In some embodiments, the invention provides methods for treating aneurodegenerative disease in a subject by administering any one of theHAT modulator compounds having formula (I) and a HDAC modulator. In someembodiments, the invention further provides methods for treating cancerin a subject by administering any one of the HAT modulator compoundshaving formula (I) and a HDAC modulator. In some embodiments, thecompound administered to a subject is any one of the compounds offormula (I) and a HDAC modulator. In some embodiments, the compound offormula (I) is any of compounds

and a HDAC modulator. In some embodiments, the HDAC modulator is a HDACinhibitor.

In some embodiments, the methods comprise administering to the subject atherapeutic amount of a HAT modulator compound and therapeutic amount aHDAC modulator compound. In some embodiments, the subject exhibitsabnormally elevated amyloid beta plaques, or elevated Tau proteinlevels, or accumulations of alpha-synuclein, or accumulations oflipofuscin, or accumulation of cleaved TARDBP (TDB-43) levels, or acombination thereof. In some embodiments, the Aβ protein depositcomprises an Aβ₄₀ isomer, an Aβ₄₂ isomer, or a combination thereof. In afurther embodiment, the subject is afflicted with Alzheimer's disease,Lewy body dementia, inclusion body myositis, Huntington's Disease,Parkinson's Disease, or cerebral amyloid angiopathy. In someembodiments, the subject is afflicted with cancer. In furtherembodiments, the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma, Bcell lymphoma, T cell lymphoma, or follicular lymphoma. In someembodiments, the B cell lymphoma is diffuse large B-cell lymphoma. Insome embodiments, the diffuse large B-cell lymphoma is a germinalcenter-derived diffuse large B cell lymphoma, an activatedB-cell-derived (ABC) diffuse large B-cell lymphoma, or non-germinalcenter diffuse large B cell lymphoma.

The dosage administered can be a therapeutically effective amount of thecomposition sufficient to result in amelioration of symptoms of aneurodegenerative disease such as, but not limited to reducing inclusionbodies (e.g., amyloid beta (Aβ) protein deposits, native andphosphorylated Tau proteins, native and phosphorylated alpha-synuclein,lipofuscin, cleaved TARDBP (TDB-43), or a combination thereof), orreducing memory loss in a subject. For example, observing at least,about a 25% reduction, at least about a 30% reduction, at least about a40% reduction, at least about a 50% reduction, at least about a 60%reduction, at least about a 70% reduction, at least about a 80%reduction, at least about a 85% reduction, at least about a 90%reduction, at least about a 95% reduction, at least about a 97%reduction, at least about a 98% reduction, or a 100% reduction ininclusion bodies or memory loss in a subject is indicative ofamelioration of symptoms of a neurodegenerative disease (for example,including, but not limited to, AD, Huntington's Disease, Parkinson'sDisease). This efficacy in reducing inclusion occurrence can be, forexample, a measure of ameliorating symptoms of a neurodegenerativedisease.

In some embodiments, the therapeutically effective amount is at leastabout 0.1 mg/kg body weight, at least about 0.25 mg/kg body weight, atleast about 0.5 mg/kg body weight, at least about 0.75 mg/kg bodyweight, at least about 1 mg/kg body weight, at least about 2 mg/kg bodyweight, at least about 3 mg/kg body weight, at least about 4 mg/kg bodyweight, at least about 5 mg/kg body weight, at least about 6 mg/kg bodyweight, at least about 7 mg/kg body weight, at least about 8 mg/kg bodyweight, at least about 9 mg/kg body weight, at least about 10 mg/kg bodyweight, at least about 15 mg/kg body weight, at least about 20 mg/kgbody weight, at least about 25 mg/kg body weight, at least about 30mg/kg body weight, at least about 40 mg/kg body weight, at least about50 mg/kg body weight, at least about 75 mg/kg body weight, at leastabout 100 mg/kg body weight, at least about 200 mg/kg body weight, atleast about 250 mg/kg body weight, at least about 300 mg/kg body weight,at least about 3500 mg/kg body weight, at least about 400 mg/kg bodyweight, at least about 450 mg/kg body weight, at least about 500 mg/kgbody weight, at least about 550 mg/kg body weight, at least about 600mg/kg body weight, at least about 650 mg/kg body weight, at least about700 mg/kg body weight, at least about 750 mg/kg body weight, at leastabout 800 mg/kg body weight, at least about 900 mg/kg body weight, or atleast about 1000 mg/kg body weight.

A HAT modulator compound and a HDAC modulator compound can beadministered to the subject one time (e.g., as a single injection ordeposition). Alternatively, a HAT modulator compound and a HDACmodulator compound can be administered once or twice daily to a subjectin need thereof for a period of from about 2 to about 28 days, or fromabout 7 to about 10 days, or from about 7 to about 15 days. It can alsobe administered once or twice daily to a subject for a period of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combinationthereof.

In some embodiments, a HAT modulator compound and a HDAC modulatorcompound are used, formulated for use and/or administered to thesubject. In some embodiments, the HAT modulator compound and the HDACmodulator are used, formulated for use and/or administered to thesubject at the same time, optionally as a composition comprising the HATmodulator compound and the HDAC modulator, or as two separate doses. Insome embodiments, the HAT modulator compound and the HDAC modulator areused, formulated for use and/or administered to the subject at differenttimes. For example, some embodiments, the HAT modulator compound is usedor administered prior to, or after the HDAC modulator. In oneembodiment, the HAT modulator is used or administered prior to, or afterthe HDAC modulator separated by a time of at least about 1 minute, 2minutes, 5 minutes, 10 minutes, 30 minutes: 45 minutes, 1 hour, 1.5hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours,12 hours 16 hours, or 24 hours. Optionally, in some embodiments the HDACmodulator issued, formulated for use and/or administered to the subjectseparated by more than about 24 hours, 36 hours, 48 hours, 3 days, 4days, 5 days, 6 days, or one week.

The dosage administered can vary depending upon known factors such asthe pharmacodynamic characteristics of the active ingredient and itsmode and route of administration; time of administration of activeingredient; age, sex, health and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency of treatmentand the effect desired; and rate of excretion.

Toxicity and therapeutic efficacy of therapeutic compositions of thepresent invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agentsthat exhibit large therapeutic indices are useful. Therapeuticcompositions that exhibit some toxic side effects can also be used.

A therapeutically effective dose of a HAT modulator compound and a HDACmodulator compound can depend upon a number of factors known to those ofordinary skill in the art. The dose(s) of a HAT modulator compound and aHDAC modulator compound, can vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the compounds are to beadministered, if applicable, and the effect which the practitionerdesires the HAT modulator compound and HDAC modulator compound to have.These amounts can be readily determined by a skilled artisan.

The HAT modulator compound and HDAC modulator compound of the inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions can comprise a HAT modulator compound(e.g., a compound of formula (I), or any of

or any combination thereof), and a pharmaceutically acceptable carrier.Other compositions can comprise a HDAC modulator compounds (e.g.romidepsin, or vorinostat) and a pharmaceutically acceptable carrier.Other compositions can comprise a HAT modulator compound (e.g., acompound of formula (I), or any of

or any combination thereof), one or more HDAC modulator compounds (e.g.romidepsin, or vorinostat) and a pharmaceutically acceptable carrier.The compositions can be administered alone or in combination with atleast one other agent, such as a stabilizing compound, which can beadministered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions can be administered to a patient alone, or incombination with other agents, drugs or hormones.

In one embodiment, the present disclosure relates to a pharmaceuticalcombination of romidepsin and

(YF2) or a pharmaceutically acceptable salt thereof. In anotherembodiment, the present disclosure relates to a pharmaceuticalcombination of romidepsin and

In one embodiment, the present disclosure relates to a pharmaceuticalcomposition comprising romidepsin,

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier or excipient. In another embodiment, the presentdisclosure relates to a pharmaceutical composition comprisingromidepsin,

and a pharmaceutically acceptable carrier or excipient.

The present invention is based, in part, on the discovery that a HATmodulator alone, or in combination with a HDAC modulator, such asromidepsin, can treat cancer or neurodegenerative disease. Thus, in someembodiments, the present invention provides methods for treating cancerin a subject in need thereof with a HAT modulator alone or incombination with a HDAC modulator such as, for example, romidepsin. Inother embodiments, the present invention provides methods for treatingneurodegenerative disease in a subject in need thereof with a HATmodulator alone or in combination with a HDAC modulator such as, forexample, romidepsin. When used in combination, the particular sequenceof administration of HAT modulator and HDAC modulator is not important.Thus, in some embodiments, the HAT modulator and HDAC modulator may beadministered at the same time. In some embodiments, the HAT modulatorand HDAC modulator may be administered at different times. In someembodiments, the HAT modulator and HDAC modulator may be administeredsequentially. In some embodiments, the HAT modulator is administeredprior to administration of the HDAC modulator. In some embodiments, theHDAC modulator is administered prior to the administration of the HATmodulator. In some embodiments, the subject is currently taking a HDACmodulator, such as romidepsin, and a HAT modulator is administered whilethe subject maintains treatment with the HDAC modulator. In someembodiments, the HAT modulator is a HAT activator. In some embodiments,the HAT modulator is a HAT inhibitor. In other embodiments, the HDACmodulator is a HDAC inhibitor. In other embodiments, the HDAC modulatoris a HDAC activator.

In some embodiments, a HAT modulator is administered together with aHDAC modulator such as, for example, romidepsin, to patients with canceror a neurodegenerative disease. In some embodiments, there is asynergistic effect between the HAT modulator and the HDAC modulator.

In some embodiments, the present invention provides a method fortreating or preventing or cancer or a neurodegenerative disease bycombined use of a HAT modulator and a HDAC modulator. In someembodiments, the methods comprise administering an amount of a HATmodulator or a pharmaceutically acceptable salt thereof in combinationwith an amount of a HDAC modulator, or a pharmaceutically acceptablesalt thereof to a subject to treat or prevent cancer or aneurodegenerative disease. In some embodiments, a synergistic effect isobserved between the HAT modulator and the HDAC modulator. In someembodiments, the HAT modulator or a pharmaceutically acceptable saltthereof and HDAC modulator, or a pharmaceutically acceptable saltthereof are administered in a therapeutically effective amount. In someembodiments, the HAT modulator, or a pharmaceutically acceptable saltthereof, along with a HDAC modulator or a pharmaceutically acceptablesalt thereof, may be a part of a pharmaceutical composition and may bedelivered alone or with other agents in combination with apharmaceutically acceptable carrier. In some embodiments, the agent isanother agent that treats cancer or neurodegenerative disease. In someembodiments, the HDAC modulator is selected from the group consisting ofromidepsin, vorinostat, belinostat, panobinostat, entinostat,mocetinostat, abexinostat, quisinostat, gavinostat and combinationsthereof. In some embodiments, the HDAC modulator is romidepsin. Inanother embodiment, the HDAC inhibitor is chidamide, resminostat,givinostat, or kevetrin.

In some embodiments, the present invention provides a method fortreating and/or preventing cancer or a neurodegenerative disease bycombined use of a HAT modulator and a HDAC modulator in subjects. Insome embodiments, there is a synergistic effect between a HAT modulatorand a HDAC modulator. Thus, in some embodiments, a HAT modulator and aHDAC modulator are administered in amounts that exhibit synergistictreatment of cancer or neurodegenerative disease. In some embodiments, aHAT modulator and a HDAC modulator are administered in amounts thatexhibit synergistic treatment and/or prevention of cancer orneurodegenerative disease.

The precise dose to be employed in the compositions will also depend onthe route of administration, and the seriousness of the affliction ordisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. In specific embodimentsof the invention, a HDAC modulator such as, for example, romidepsin canbe administered by known methods and dose ranges.

In some embodiments, the doses are used when a HAT modulator isadministered alone. In some embodiments, the doses are used when a HATmodulator is administered in combination with a HDAC modulator. In someembodiments, the doses are administered orally. In some embodiments, thedoses are administered intravenously.

The HAT modulator and HDAC modulator (or pharmaceutically acceptablesalts of either HAT modulator and HDAC modulator or both) can beadministered at different times or at the same time.

In some embodiments, the compositions of the invention comprise a HATmodulator and a HDAC modulator. In some embodiments, a HAT modulator anda HDAC modulator are present together in a single dosage form such as,for example, an oral dosage form. In some embodiments, a HAT modulatorand a HDAC modulator are present together in a single dosage form suchas, for example, an intravenous dosage form. In some embodiments, a HATmodulator and a HDAC modulator are administered separately. In someembodiments, a HAT modulator is present together in a single dosage formsuch as, for example, an oral dosage form. In some embodiments, a HATmodulator is present together in a single dosage form such as, forexample, an intravenous dosage form. In some embodiments, a HDACmodulator is present together in a single dosage form such as, forexample, an oral dosage form. In some embodiments, a HDAC modulator ispresent together in a single dosage form such as, for example, anintravenous dosage form.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

Any of the therapeutic applications described herein can be applied toany subject in need of such therapy, including, for example, a mammalsuch as a mouse, rat, dog, cat, cow, horse, rabbit, monkey, pig, sheep,goat, or human. In some embodiments, the subject is mouse, rat, monkey,dog or human. In some embodiments, the subject is a mouse, monkey orhuman. In some embodiments, the subject is a human.

A pharmaceutical composition of the invention can be formulated to becompatible with its intended route of administration. Exemplary routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Theinjectable composition should be sterile and should be fluid to theextent that easy syringability exists. It should also be stable underthe conditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyethylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal. In manyembodiments, it can be useful to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and/or sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the HATmodulator compound and the HDAC modulator compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated herein.In the case of sterile powders for the preparation of sterile injectablesolutions, examples of useful preparation methods are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Itwill be recognized that one or more features of any embodiments oraspects disclosed herein can be combined and/or rearranged within thescope of the invention to produce further embodiments that are alsowithin the scope of the invention.

As will be apparent to one of ordinary skill in the art from a readingof this disclosure, the embodiments of the present disclosure can beembodied in forms other than those specifically disclosed above. Theparticular embodiments described herein are, therefore, to be consideredas illustrative and not restrictive. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific embodimentsdescribed herein. The scope of the invention is as set forth in theappended claims and equivalents thereof, rather than being limited tothe examples contained in the foregoing description.

The invention is further described by the following non-limitingExamples. Examples of mouse models for neurodegenerative diseases,including, Alzheimer's Disease, Huntington's Disease and Parkinson'sDisease are described in WO 2011/072243 and WO 2012/088420, eachincorporated by reference herein in its entirety.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1

Methyl 2-methoxy-6-methylbenzoate, 4

A vigorously mixture of 1-methoxy-2,3-dimethylbenzene (1, 1.34 mL),Copper (II) Sulfate pentahydrate (2.5 g) and potassium peroxodisulfate(8.1 g) in acetonitrile/water 1:1 (70 mL) was heated at reflux for 6 h.The reaction was cooled to room temperature and was extracted withdichloromethane (3 times). The organic layer was dried over Na₂SO₄,filtered and evaporated to produce the desired liquid product (2)suitable for further reaction without purification. A solution of 2(1.50 g) and sulfamic acid (1.30 g) in water (22.5 mL) and THE (11.2 mL)was stirred at room temperature and after 5 min a solution of NaClO₂(1.180 g) in water (5 mL) was added. The reaction was stirred at roomtemperature for 1 h and then was extracted with ethyl acetate. Theorganic layer was separated and extracted with NaOH 1M. The aqueoussolution was acidified with HCl 6N and extracted with dichloromethane.The organic layer was dried over Na₂SO₄, filtered and evaporated toobtain the solid product (3, 1.071 g). Thionyl chloride (0.55 mL) wasadded dropwise to a solution of 3 (83 mg) in methanol (0.5 mL) and thereaction was refluxed for 10 h. The methanol was removed by rotaryevaporator and the residue was dilutes with water and extracted withethyl acetate. The organic layer was washed with saturated solution ofNaHCO₃ and then separated, dried over Na₂SO₄, filtered and evaporated toproduce the crude product. Purification by flash chromatography gave thedesired product as colorless oil (70 mg, yield 80%). C₁₀H₁₂O₃, MS-ES:[M+H]⁺=181 m/z. ¹H-NMR: (CDCl₃, 300 MHz) δ 2.29 (s, 3H, —CH₃), 3.83 (s,3H, —OCH₃), 3.92 (s, 3H, —C(═O)OCH₃), 6.76 (d, 1H, Jo=8.7 Hz, H-5), 6.80(dd, 1H, Jo=7.8 Hz, Jm=0.6 Hz, H-3), 7.25 (t, 1H, Jo=7.5 Hz, Jm=8.4 Hz,H-4).

methyl 2-(bromomethyl)-6-methoxybenzoate, 5

N-Bromosuccinimide (250 mg) and catalytic amount of2,2′-Azobis(2-methylpropionitrile) was added to a solution of 4 (252 mg)in carbon tetrachloride (3.5 mL). The reaction was heated to reflux inthe present of visible light for 6 h. After cooling to room temperaturethe reaction was filtered and the filtrate was evaporated. The residuewas diluted with water and extracted with diethyl ether. The organiclayer was dried over Na₂SO₄, filtered and evaporated. The obtainedresidue was purified by flash chromatography to produce the desiredproduct (195 mg, yield 60%). C₁₀H₁₁BrO₃, MS-ES: [M+H]⁺=259 m/z,[M+H]⁺+2=261 m/z. ¹H-NMR: (CDCl₃, 300 MHz) δ 3.83 (s, 3H, —OCH₃), 3.94(s, 3H, —C(═O)OCH₃), 4.48 (s, 2H, —CH₂Br), 6.89 (d, 1H, Jo=8.4 Hz, H-5),7.00 (d, 1H, Jo=7.5 Hz, H-3), 7.33 (t, 1H, Jo=8.1 Hz, H-4).

2-(4-chloro-3-(trifluoromethyl)phenyl)-7-methoxyisoindoline-1-one, 6

A solution of 5 (130 mg), 4-chloro-3-(trifluoromethyl)aniline (98 mg),triethylamine (105 μL) and K₂CO₃ (10 mg) was refluxed in acetone (1 mL)for 6 h. The reaction was extracted with ethyl acetate and water. Theorganic layer was dried over Na₂SO₄, filtered and evaporated. The crudeproduct was purified by flash chromatography (dichloromethane/ethylacetate, 9.5:0.5) to obtain the desired product (65 mg, yield 80%).C₁₆H₁₁ClF₃NO₂, MS-ES: [M+H]⁺=342 m/z. ¹H-NMR: (CDCl₃, 300 MHz) δ 4.01(s, 3H, —OCH₃), 4.80 (s, 2H, —CH₂), 6.95 (d, 1H, Jo=8.1 Hz, H-4), 7.08(d, 1H, Jo=7.2 Hz, H-6), 7.52 (d, 1H, Jo=9.0 Hz, H-5′), 7.56 (t, 1H,Jo=8.0 Hz, H-5), 8.06 (dd, 1H, Jo=9.2 Hz, Jm=2.7 Hz, H-6′), 8.23 (d, 1H,Jm=2.7 Hz, H-2′).

2-(4-chloro-3-(trifluoromethyl)phenyl)-7-hydroxyisoindolin-1-one, RP23

Boron tribromide (1M in dichloromethane, 0.4 mL) was added dropwise to asolution of 6 (34 mg) in 1 mL of dichloromethane at −20° C. The reactionwas stirred for 30 min at −20° C. and overnight at 0° C. The mixture waspoured into ice-cold water and stirred for 30 min at room temperature.The product was filtered and washed with water to obtain the desiredproduct (30 mg, yield: 89%). C₁₅H₉ClF₃NO₂, MS-ES: [M+H]⁺=328 m/z.¹H-NMR: (CDCl₃, 300 MHz) δ 4.86 (s, 2H, —CH₂), 6.93 (dd, 1H, Jo=8.7 Hz,Jm=0.6 Hz, H-4), 7.02 (dd, 1H, Jo=7.8 Hz, Jm=0.6 Hz, H-6), 7.51 (t, 1H,Jo=8.0 Hz, H-5), 7.55 (d, 1H, Jo=9.0 Hz, H-5′), 8.04 (dd, 1H, Jo=8.7 Hz,Jm=2.7 Hz, H-6′), 8.14 (d, 1H, Jm=2.7, H-2′), 8.57 (s, 1H, sc. D₂O, OH).

Example 2

2-ethoxy-6-hydroxybenzoic Acid, 8

NaOH 1N (6 mL) was added dropwise to a solution of ethyl2-ethoxy-6-hydroxybenzoate (7, 1 g) in ethanol (3 mL). The reaction wasrefluxed for 2 h and then concentrated and extracted with water anddichloromethane (3 times). The aqueous solution was separated andacidified to pH=1 to obtain the precipitation of the product (660 mg,yield: 76%), which was filtrated and washed with water. C₉H₁₀O₄, MS-ES:[M+H]⁺=183 m/z. ¹H-NMR: (CDCl₃, 300 MHz) δ 1.57 (t, 3H, Jv=7.2 Hz, —CH₂CH₃ ), 4.32 (q, 2H, Jv=7.2 Hz, —CH₂ CH₃), 6.47 (d, 1H, Jo=8.4 Hz, H-5),6.71 (d, 1H, Jo=8.4 hz, H-3), 7.39 (t, 1H, Jo=8.4 Hz, H-4), 11.60 (s,1H, C(═O)OH), 12.16 (s, 1H, OH).

N-(4-chloro-3-(trifluoromethyl)phenyl)-2-ethoxy-6-hydroxybenzamide, 9

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (900 mg)was added gradually to a solution of 8 (660 mg) and4-chloro-3-(trifluoromethyl)aniline (780 mg) in dichloromethane (5 mL)at 0° C. The reaction was stirred at room temperature overnight thenfiltered and the precipitate was crystallized from methanol (608 mg,yield: 67%). C₁₆H₁₃ClF₃NO₃, MS-ES: [M+H]⁺=360 m/z. ¹H-NMR: (CDCl₃, 300MHz) δ 1.65 (t, 3H, Jv=6.9 Hz, —CH₂ CH₃ ), 4.27 (q, 2H, Jv=6.9 Hz, —CH₂CH₃), 6.44 (d, 1H, Jo=8.4 Hz, H-5), 6.67 (d, 1H, Jo=8.4 Hz, H-3), 7.32(t, 1H, Jo=8.4 Hz, H-4), 7.48 (d, 1H, Jo=8.7 Hz, H-5′), 7.77 (dd, 1H,Jo=8.7 Hz, Jm=2.4 Hz, H-6′), 7.91 (d, 1H, Jm=2.1 Hz, H-2′), 10.66 (s,1H, —C(═O)NH), 13.29 (s, 1H, OH).

2-(benzyloxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)-6-ethoxybenzamide,RP62

Benzyl bromide (24 μL) was added to a suspension of 9 (70 mg) and K₂CO₃(36 mg) in DMF (1.5 mL). The reaction was stirred at room temperatureand after 18 h the solvent was evaporated under vacuum and the residuepartitioned between saturated aqueous solution of NaHCO₃ and ethylacetate. The organic layer was separated, dried over Na₂SO₄, filteredand evaporated. Purification by flash chromatography (Hexane/Ethylacetate, 8:2) gave the desired product (50 mg, yield: 60%).C₂₃H₁₉ClF₃NO₃, MS-ES: [M−H]⁻=448 m/z, [M+H]⁺=450 m/z. ¹H-NMR: (DMSO-d₆,400 MHz) δ 1.20 (t, 3H, Jv=6.9 Hz, —CH₂-CH₃ ), 4.02 (q, 2H, Jv=6.9 Hz,—CH₂ —CH₃), 5.10 (s, 2H, CH₂-Ph), 6.70 (d, 1H, Jo=7.6 Hz, H-3), 6.74 (d,1H, Jo=8.4 Hz, H-5), 7.12-7.34 (m, 6H), 7.49 (d, 1H, Jo=9.2 Hz, H-5′),7.90 (dd, 1H, Jo=8.8 Hz, Jm=2.4 Hz, H-6′), 8.25 (d, 1H, Jm=2.4 Hz,H-2′), 10.67 (s, 1H, NH).

2-(benzyloxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)-6-ethoxy-N-methylbenzamide,RP65

Iodomethane (20 μL) was added to suspension of RP62 (70 mg) and NaH (60%oil disp., 9 mg) in THE (2 mL) at room temperature. The reaction wasstirred and after 12 h the solvent was evaporated and the residue wasdiluted with HCl 1M and extracted with dichloromethane (2 times). Theorganic layer was dried over Na₂SO₄, filtered and evaporated obtainingthe desired product (80 mg, yield: 30%). C₂₄H₂₁ClF₃NO₃, MS-ES:[M+H]⁺=464 m/z. ¹H-NMR: (CDCl₃, 400 MHz) δ 1.40 (dt, 3H, Jv=7.2 Hz, —CH₂CH₃ ), 3.40 (s, 3H, N—CH₃), 3.90 (dd, 1H, Jv=7.2 Hz, —CH₂ CH₃), 4.03(dd, 1H, Jv=7.2, —CH₂ CH₃), 4.90 (d, 1H, Jv=12 Hz, CH₂-Ph), 5.05 (d, 1H,Jv=12 Hz, CH₂-Ph), 6.33 (dd, 2H, Jo=7.6 Hz, H-3 and H-5), 7.04 (t, 1H,Jo=8.8 Hz, H-4), 7.11 (dd, 1H, Jm=2.8 Hz, Jo=9.0 Hz, H-6′), 7.18 (d, 1H,Jo=8.8 Hz, H-5′), 7.26-7.38 (m, 5H), 7.46 (d, 1H, Jm=2.4 Hz, H-2′).

N-(4-chloro-3-(trifluoromethyl)phenyl)-2-ethoxy-6-hydroxy-N-methylbenzamide,RP71

A solution of RP65 (80 mg) in ethyl acetate was hydrogenated atatmospheric pressure over 10% Pd/C (19 mg) for 24 h. The mixture wasfiltered and the filtrate was evaporated to give the product (60 mg,yield: 94%). C₁₇H₁₅ClF₃NO₃. ¹H-NMR: (CDCl₃, 400 MHz) δ 1.20-1.22 (m, 3H,—CH₂ CH₃ ), 3.42 (s, 3H, N—CH₃), 3.54-3.60 (m, 2H, —CH₂ CH₃), 6.06 (d,1H, Jo=8.4 Hz, H-3), 6.49 (d, 1H, Jo=8.4 Hz, H-5), 7.02-7.06 (m, 2H,H-6′ and H-5′), 7.02-7.08 (m, 1H, H4), 7.40-7.42 (m, 1H, H-2′), 7.68 (s,1H, OH).

Example 3

2-bromo-6-fluorobenzoic Acid, 11

A solution 2-bromo-6-fluorobenzonitrile 10 in KOH 1M (25 mL) was stirredto reflux for 2 day. The reaction was cooled to room temperature and theHCl concentrate was added to pH=2-3. The aqueous solution was extractedwith ethyl acetate (3 times). The organic layer was separated, dried andevaporated to obtain the desired product (126 mg, yield: 95%).C₇H₄BrFO₂, MS-ES: [M−H]⁻=218 m/z. ¹H-NMR: (CDCl₃, 400 MHz) δ 7.14 (t,1H, Jo=8.4 Hz, H-4), 7.29-7.35 (m, 1H, H-3), 7.45 (d, 1H, H-5).

2-bromo-N-(4-chloro-3-(trifluoromethyl)phenyl)-6-fluorobenzamide, RP106

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC HCl,74 mg) was added to a solution of 11 (65 mg) in dichloromethane (0.5 mL)at 0° C., and then 4-chloro-3-(trifluoromethyl)aniline (64 mg) wasadded. The solution was stirred at room temperature for 24 h. Thesolvent was evaporated and the residue was crystallized from methanol(55 mg, yield: 47%). C₁₄H₇BrClF₄NO, MS-ES: [M−H]⁻=394 m/z. ¹H-NMR:(CDCl₃, 400 MHz) δ 7.10 (t, 1H, Jo=8.4 Hz, H-4), 7.30-7.40 (m, 3H, H-3,H-6′ and H-5′), 7.02-7.08 (m, 1H, H-5), 7.40-7.42 (m, 1H, H-2′), 7.60(s, 1H, NH).

Example 4

2-(4-chloro-3-(trifluoromethyl)benzyl)-4-hydroxyisoindoline-1,3-dione,RP74

A solution of 12 (100 mg) and 4-chloro-3-(trifluoromethyl)benzylamine(111 μL) in acetic acid (3 mL) was heated to reflux for 2 h. The solventwas evaporated and the residue was extracted with dichloromethane andwater. The organic layer was dried over Na₂SO₄, filtered and evaporated.The crude product was purified by flash chromatography(dichloromethane/methanol, 9.9:0.1)(89 mg, yield: 45%). C₁₆H₉ClF₃NO₃,MS-ES: [M−H]⁻=354 m/z. ¹H-NMR: (CDCl₃, 400 MHz) 4.79 (s, 2H, CH₂-Ph),7.16 (d, 1H, Jo=8.0 Hz, H-7), 7.38 (d, 1H, Jo=6.8 Hz, H-5), 7.45 (d, 1H,Jo=8.4 Hz, H-5′), 7.52 (dd, 1H, Jo=7.6 Hz, Jm=2.0 Hz, H-6′), 7.51-7.53(br s, 1H, OH), 7.58 (dd, 1H, Jo=8.8 Hz, Jo=8.4 Hz, H-6), 7.72 (d, 1H,Jm=2.0 Hz, H-2′).

2-(4-chloro-3-(trifluoromethyl)benzyl)-4-(2-(dimethylamino)ethoxy)isoindoline-1,3-dione,RP78

A suspension of RP74 (89 mg), 2-Chloro-N,N-dimethylethylaminehydrochloride (40 mg), and K₂CO₃ (86 mg) in DMF (2 mL) was stirred at80° C. for 24 h. The reaction mixture was diluted with dichloromethaneand washed with water (3 times). The organic layer was dried overNa₂SO₄, filtered and evaporated. The crude product was purified by flashchromatography (methanol/ethyl acetate, 5:5) to provide the desiredproduct (40 mg, yield: 40%). C₂₀H₁₈ClF₃N₂O₃, MS-ES: [M+H]⁺=427 m/z.¹H-NMR: (CDCl₃, 400 MHz) 2.37 (s, 6H, —N(CH₃)₂), 2.83 (t, 2H, Jv=6.0 Hz,—OCH₂ CH₂ —N—), 4.26 (t, 2H, Jv=6.0 Hz, —OCH₂ CH₂—N—), 4.78 (s, 2H,CH₂-Ph), 7.18 (d, 1H, Jo=8.0 Hz, H-7), 7.42 (d, 1H, Jo=8.4 Hz, H-5),7.43 (d, 1H, Jo=6.8 Hz, H-5′), 7.53 (dd, 1H, Jo=8.4 Hz, Jo=8.0 Hz,Jm=2.0 Hz, H-6′), 7.63 (dd, 1H, Jo=8.8 Hz, Jo=8.4 Hz, H-6), 7.72 (d, 1H,Jm=2.0 Hz, H-2′).

2-(4-chloro-3-(trifluoromethyl)benzyl)-4-ethoxyisoindoline-1,3-dione,RP79

A suspension of RP74 (95 mg), bromoethane (23 μL), and K₂CO₃ (93 mg) inDMF (2.5 mL) was stirred at 80° C. for 24 h. The reaction mixture wasdiluted with dichloromethane and washed with water (3 times). Theorganic layer was dried over Na₂SO₄, filtered and evaporated. The crudeproduct was purified by flash chromatography (hexane/ethyl acetate, 6:4)to provide the desired product (95 mg, yield: 93%). C₁₈H₁₃ClF₃NO₃,MS-ES: [M+H]⁺=384 m/z. ¹H-NMR: (CDCl₃, 400 MHz) δ 1.50 (t, 3H, Jv=7.2Hz, —CH₂ CH₃ ), 4.24 (q, 2H, Jv=7.2 Hz, —CH₂ CH₃), 4.78 (s, 2H, CH₂-Ph),7.16 (d, 1H, Jo=8.0 Hz, H-7), 7.40 (d, 1H, Jo=7.2 Hz, H-5), 7.41 (d, 1H,Jo=8.4 Hz, H-5′), 7.54 (dd, 1H, Jo=8.4 Hz, Jm=2.0 Hz, H-6′), 7.62 (dd,1H, Jo=7.2 Hz, H-6), 7.72 (d, 1H, Jm=2.0 Hz, H-2′).

Example 5

N-(4-chloro-3-(trifluoromethyl)phenyl)-2-(2-(dimethylamino)ethoxy)-6-ethoxybenzamide,13

Diisopropyl azodicarboxylate (128 μL) was added to a solution of 9,2-Dimethylaminoethanol (65 μL) and triphenylphosphine (170 mg) in THF(2.5 mL) at 0° C. The solution was stirred at room temperatureovernight. The solvent was evaporated and the residue was diluted inethyl acetate and washed with water and brine (3 times). The organiclayer was dried over Na₂SO₄, filtered and evaporated. Purification byflash chromatography (dichloromethane/methanol, 9.4:0.6) gave thedesired colorless oil. C₂₀H₂₂ClF₃N₂O₃, MS-ES: [M+H]⁺=431 m/z. ¹H-NMR:(CDCl₃, 400 MHz) δ 1.39 (t, 3H, Jv=6.9 Hz, O—CH₂ CH₃ ), 2.25 (s, 6H,N(CH₃)₂), 2.65 (t, 2H, Jv=5.4 Hz, —OCH₂ CH₂ N—), 4.09 (q, 2H, Jv=6.9 Hz,—OCH₂ CH₃), 4.19 (t, 2H, Jv=5.4 Hz, —OCH₂ CH₂N—), 6.59 (d, 1H, Jo=8.7Hz, H-3), 6.60 (d, 1H, Jo=8.7 Hz, H-5), 7.28 (t, 1H, Jo=8.4 Hz, Jo=8.7Hz, H-4), 7.46 (d, 1H, Jo=8.4 Hz, H-5′), 7.80 (s, 1H, H-2′), 7.98 (d,1H, Jo=8.1, H-6′), 8.71 (s, 1H, NH).

2-(2-((4-chloro-3-(trifluoromethyl)phenyl)carbamoyl)-3-ethoxyphenoxy)-N,N,N-trimethylethane-1-aminiumiodide, RP17

Iodomethane (21 μL) was added to a solution of 13 (86 mg) in diethylether (1.2 mL) and the reaction was stirred at room temperatureovernight. The white precipitate was collected by filtration and driedunder vacuum to give the desired product (75 mg, yield: 65%).C₂₁H₂₅ClF₃IN₂O₃, MS-ESI: [M]⁺=445 m/z. ¹H-NMR: (DMSO-d₆, 400 MHz) δ 1.22(t, 3H, Jv=7.1 Hz, —OCH₂ CH₃ ), 3.04 (s, 9H, —N(CH₃)₃), 3.64 (m, 2H,—OCH₂ CH₂ N), 4.06 (q, 2H, Jv=6.9 Hz, —OCH₂ CH₃), 4.45 (m, 2H, —OCH₂CH₂N), 6.79 (dd, 2H, Jo=8.7 Hz, Jm=2.0 Hz, H-4 and H-6), 7.39 (t, 1H,Jo=8.4 Hz, H-5), 7.67 (d, 1H, Jo=8.7 Hz, H-5′), 7.86 (d, 1H, Jo=8.1 Hz,H-6′), 8.27 (d, 1H, Jm=2.4 Hz, H-2′), 10.67 (s, 1H, NH).

Example 6

Isatoic anhydride was reacted with 4-chloro-3-trifluoromethyl aniline toprovide RP 95:

Example 7

2-bromo benzoic acid was reacted with 4-chloro-3-trifluoromethyl anilineto provide RP 101. RP 102 was obtained via reaction of RP101 with N,N-dimethylethane diamine.

Example 8

In vitro measurements of pCAF enzymatic activity were performed withradioassay in which active pCAF (recombinant protein expressed in E.Coli, Millipore) was used to acetylate core histones (chickenerythrocyte histones, Millipore) in vitro with tracer levels oftritiated acetyl-CoA (Perkin Elmer) as the acetyl donor. Results areshown in FIG. 21.

Example 9

Histone Acetyl Transferase Activators as a Therapeutic Agent for Cancerand Inflammatory Diseases.

Described herein is a group of chemical entities which activate histoneacetyl transferases leading to acetylation of p53 and induction of p21,at concentrations between 1-10 uM. The name of the chemical identitiesthat were tested are: YF2, JF1, JF2, JF3, JF4, JF5, JF7, JF8, JF9, JF10,JF16, JF18, RP4, RP7, RP23, RP52, RP58, RP59, RP72, RP78, RP79, RP102.See FIGS. 1-4 for the structures and scheme of synthesis ofrepresentative compounds. Syntheses of other chemical entities aredescribed, for example, in WO 2011/072243; WO 2012/088420; and U.S.Patent Publication No. 2013/0121919; each herein incorporated byreference in its entirety. Treatment of lymphoma cell lines with HATactivators as a single agent has a modest impact on cell viability over24-72 hours. However, the combination of HAT inducer (RP52, RP59, butnot RP14) with a HDAC inhibitor such as romidepsin produces synergycoefficients lower as low as 0.14 starting at 48 hours (synergycoefficients less than 1 indicate synergy) and reduced cell viability(FIGS. 5-18).

Luminetric assays for synergy between romidepsin and RP14 evaluated indifferent cell lines (FIGS. 5-7): Cells were untreated (bar labeled“c”); treated with 1.5 nM Romidepsin alone (bar labeled “1.5 R”);treated with RP14 alone at increasing concentrations from 1-10 μm (barlabeled 1, 2, 4, 6, 8, or 10); or treated with 1.5 nM Romidepsin incombination with RP14 at increasing concentrations from 1-10 μm (barlabeled R+1, R+2, R+4, R+6, R+8, or R+10).

Luminetric assays for synergy between romidepsin and RP52 evaluated indifferent cell lines (FIGS. 8-10): Cells were untreated (bar labeled“c”); treated with 1.5 nM Romidepsin alone (bar labeled “1.5 R”);treated with RP52 alone at increasing concentrations from 1-10 μm (barlabeled 1, 2, 4, 6, 8, or 10); or treated with 1.5 nM Romidepsin incombination with RP52 at increasing concentrations from 1-10 μm (barlabeled R+1, R+2, R+4, R+6, R+8, or R+10).

Luminetric assays for synergy between romidepsin and RP59 evaluated indifferent cell lines (FIGS. 11-13): Cells were untreated (bar labeled“c”); treated with 1.5 nM Romidepsin alone (bar labeled “1.5 R”);treated with RP59 alone at increasing concentrations from 1-10 μm (barlabeled 1, 2, 4, 6, 8, or 10); or treated with 1.5 nM Romidepsin incombination with RP59 at increasing concentrations from 1-10 μm (barlabeled R+1, R+2, R+4, R+6, R+8, or R+10).

Luminetric assays for synergy between romidepsin and different HATactivators (RP14, RP52, RP72, JF2) evaluated in different cell lines(FIGS. 14-15): Cells were untreated (bar labeled “H9”); treated with 2.5nM Romidepsin alone (bar labeled “R 2.5 nM”); treated with HAT activatoralone (bar labeled RP14, RP52, RP72, JF2); or treated with 2.5 nMRomidepsin in combination with HAT activator (bar labeled R+RP14,R+RP52, R+RP72, R+JF2).

Luminetric assays for synergy between romidepsin and RP14 evaluated indifferent cell lines (FIGS. 16-17): Cells were untreated (bar labeled“H9”); treated with 2 nM Romidepsin alone (bar labeled “R2 nM”); treatedwith RP14 alone at increasing concentrations from 0.1-10 μm (bar labeledRP14 0.1 μM, RP14 1 μM, RP14 2.5 μM, RP14 5 μM, RP14 10 μM); or treatedwith 2 nM Romidepsin in combination with RP14 at increasingconcentrations from 0.1-10 μm (bar labeled R+RP14 0.1 μM, R+RP14 1 μM,R+RP14 2.5 μM, R+RP14 5 μM, R+RP14 10 μM).

Luminetric assays for synergy between romidepsin and RP72 evaluated indifferent cell lines (FIG. 18): Cells were untreated (bar labeled “HH”);treated with 1.25 nM Romidepsin alone (bar labeled “R1.25 nM”); treatedwith RP72 alone at increasing concentrations from 0.1-10 μm (bar labeledRP72 0.1 μM, RP72 1 μM, RP72 2.5 μM, RP72 5 μM, RP72 10 μM); or treatedwith 1.25 nM Romidepsin in combination with RP72 at increasingconcentrations from 0.1-10 μm (bar labeled R+RP72 0.1 μM, R+RP72 1 μM,R+RP72 2.5 μM, R+RP72 5 μM, R+RP72 10 μM).

Treatment of diffuse large B-cell lymphoma cell lines (OCI-Ly1 andSu-DHL6) with RP52 lead to increased acetylation of p53 and induction ofp21, a key regulator of the cell cycle (FIGS. 19A-B). Cells were treatedwith Romidepsin 1.5 nM, RP52 5 uM or the combination for 24 hours thenlysed with RIPA buffer. Levels of total p53, acetylated p53, and p21were detected by Western Blot analysis. Antibodies used wereanti-acetyl-p53 (Santa Cruz), anti-p53 DO-1 (Abcam), anti-p21 (CellSignaling).

The pharmaco-modulation of key tumor suppressors induced by HATactivators and their potent synergy with HDAC inhibitors give credenceto the further development of these new chemical entities for thetreatment of cancers and inflammatory disorders.

FIGS. 20A-D show the concentration-effect relationship for 21 HATactivator compounds in a panel of non-Hodgkin's lymphoma cell lines at48 hours. Cell lines tested were germinal center-derived (GC) diffuselarge B-cell lymphoma (DLBCL) cell lines (Ly1, Ly7, activatedB-cell-derived (ABC) diffuse large B-cell lymphoma (DLBCL) cell lines(Ly10, SU-DHL2), or T-cell lymphoma cell lines (HH, H9). The cells wereplated at a concentration of 3×10⁵ cells/mL in a volume of 1 mL percondition and viability was measured by the cell titer glo assay. Cellswere treated with the HAT activators at two concentrations (2.5 uM and 5uM) for evaluation of the single agent activity. Synergy for 21 HATactivator compounds (YF2, JF1, JF3, JF4, JF5, JF7, JF8, JF9, JF10, JF16,JF18, RP14, RP17, RP23, RP52, RP58, RP59, RP72, RP78, RP79, RP102) withthe pan-class histone deacetylase inhibitor, romidepsin, was evaluatedat the inhibitory concentrations 10% (1 nM) and 20% (1.5 nM). Synergycoefficients were calculated as the relative risk ratio (RRR).Combinations yielding values RRR<1 are synergistic whereas thoseyielding RRR>1 are antagonistic, and RRR=1 are additive. FIG. 20A showsthe percentage viability of cells treated with HAT activators YF2, JF1,JF3, JF4, JF5, JF7, JF8, JF9, JF10, JF16, JF18 as single agents or incombination with romidepsin at 48 hours. FIG. 20B shows the percentageviability of cells treated with HAT activators RP14, RP17, RP23, RP52,RP58, RP59, RP72, RP78, RP79, RP102 as single agents or in combinationwith romidepsin at 48 hours. FIG. 20C shows the synergy coefficientscalculated as the relative risk ratio (RRR) for cells treated with HATactivators YF2, JF1, JF3, JF4, JF5, JF7, JF8, JF9, JF10, JF16, JF18 assingle agents or in combination with romidepsin at 48 hours. FIG. 20Dshows the synergy coefficients calculated as the relative risk ratio(RRR) for cells treated with HAT activators RP14, RP17, RP23, RP52,RP58, RP59, RP72, RP78, RP79, RP102 as single agents or in combinationwith romidepsin at 48 hours.

FIGS. 22A-E show the synergy effect of JF1, a HAT activator compound,with romidepsin (a pan-class HDAC inhibitor) in B-cell lymphoma celllines. Synergy between JF1 and romidepsin was evaluated in Pfeiffer(EP300 wt/CREBBP mut) and SUDHL-10 cells (EP300 mut/CREBBP A). The cellswere exposed to increasing concentration of JF1 and romidepsin alone andin combinations as indicated in FIGS. 22A and 22B. Excess Over Bliss wascalculated. Drug synergy will be confirmed by Excess over bliss (EOB).Bliss is calculated by the following formula: X=(A+B)−(A*B); where Xdesignates the combined response for the two single compounds witheffects (inhibition) A and B. The difference between Bliss and observedgrowth inhibition (Y) induced by combinations of drugs A and B at thesame dose is the termed EOB (EOB=Y−X) 18,19. An EOB>10 connotes synergy.Strong synergy was observed in SUDHL-10, whereas weak synergy was seenin Pfeiffer. Synergy effect of JF1 and romidepsin was also assessed in apanel of B-cell lymphoma cell lines (N=5) (FIGS. 22C-22E). Cells weretreated by different concentrations of JF1 and romidepsin for 48 hrs(FIG. 22C) and 72 hrs (FIG. 22D) and an Excess Over Bliss was calculatedfor both time points for all five cell lines. SUDHL-6 and Pfeiffer(Bold) were treated 2, 2.5, 3 nM JF1, whereas the rest of cell lineswere treated with 2, 3, 4 μM JF1. The synergy of JF1 and romidepsin ineach cell line and at each time point is indicated in FIG. 22E. Synergyis defined by an Excess Over Bliss of 10. Cell lines with EP300mutations have stronger synergy than EP300 wildtype (wt) cell lines.Genetic status of the cell lines disclosed in FIGS. 22A-22E are asfollows: Pfeiffer: EP300 wt/CREBBP mis mut; SUDHL-10: EP300 mismut/CREBBP trunc mut; SUDHL-6: EP300 mis mut/CREBBP trunc mut; RIVA:EP300 wt/CREBBP trunc mut; Ly-7: EP300 wt/CREBBP wt.

FIG. 23 shows the synergy effect of JF1 (a HAT activator compound) withromidepsin (a pan-class HDAC inhibitor) in ten B-cell lymphoma celllines and four T-cell lymphoma cell lines. Cells were exposed toincreasing concentrations of JF1 and romidepsin in various combinationsfor 72 hrs and Excess Over Bliss (EOB) was calculated. The dataillustrates that strong synergy was found in 5 out of 14 cell lines(36%) treated with JF1 in combination with romidepsin, as defined by anEOB of at least 20

FIG. 24 shows the synergy effect of YF2 (a HAT activator compound) withromidepsin (a pan-class HDAC inhibitor) in seven B-cell lymphoma celllines. Cells were exposed to increasing concentrations of YF2 andromidepsin in various combinations for 72 hrs and Excess Over Bliss(EOB) was calculated. The data illustrates that strong synergy was foundin 6 out of 7 cell lines (86%) treated with YF2 in combination withromidepsin, as defined by an EOB of at least 20. Only in Pfeiffer cellsdid the drug combination fail to show a strong response.

FIGS. 25A-B compare the synergy effects that HAT-activators JF1 and YF2in combination with romidepsin have on histone acetylation in B-celllymphoma cell lines, as quantified by mass spectrometry. In theseexperiments cells were seeded at 5,000 cells/mL and exposed to YF2 (6uM), JF1 (3 uM) and romidepsin (1.5 nM) alone and in combinations for 48hours. Following treatment, SUDHL-6 (EP300 mut/CREBBP mut, FIG. 25A) andSUDHL-10 cells (EP300 mut/CREBBP mut, FIG. 25B) were harvested forhistone extraction.

Treatment of SUDHL-6 cells with romidepsin, JF1, YF2, JF1/romidepsin,and YF2/romidepsin at the aforementioned concentrations resulted in theextraction of five lysine-acetylated histones: H2A:K5AC, H3.1:K27AC,H3.3:K27AC, H3:K9AC, and H3:K18AC. For each, a synergy effect,calculated as a fold change of treatment vs. control, was observedshowing an increase in histone acetylation for JF1/romidepsin andYF2/romidepsin compared to individual treatment of the cells with eitherJF1, YF2, or romidepsin alone (FIG. 25A). JF1 or YF2 in combination withromidepsin induced the global acetylation of histones compared withsingle agent effects. In the case of histone H3:K9AC, both combinationtreatments resulted in a 10-fold increase in acetylation vs. control,which amounted to about a 2-fold increase vs. romidepsin and an 8-foldincrease vs. YF2 or JF1 alone.

The synergy trend for the SUDCL-6 cell line is evident for SUDCL-10 aswell (FIG. 25B). In the same manner as described above, SUDHL-10 cellswere treated separately with romidepsin, JF1, YF2, JF1/romidepsin, andYF2/romidepsin and the fold change in histone acetylation was measured.JF1 or YF2 in combination with romidepsin induced the global acetylationof histones compared with single agent effects. While the JF1/romidepsincombination exhibited a slightly superior synergy effect overYF2/romidepsin when compared to control and romidepsin alone, bothcombinations demonstrated a synergy effect across all the histonesexamined in the study. The maximum effect found for H3:K18AC was about a1.8-fold increase for the JF1/romidepsin combination over romidepsinalone, but similar levels (about 1.6-fold enhancements) were observedfor other histones as well. YF2/romidepsin combinations exhibited amaximum synergy effect of about 1.6-fold for H3:K9AC.

FIG. 26 provides a Western Blot showing the effect of JF1 and YF2 aloneand in combination with romidepsin on histone acetylation in HAT-mutatedcells lines. Cells were exposed to YF2 and romidepsin alone and incombinations for 48 hours in SUDHL-6 and SUDHL-10 (EP300 mut/CREBBPmut). The synergistic effect of YF2 on histone acetylation was evaluatedby Western Blot with histone H3 serving as loading control. YF2 incombination with romidepsin induced the global acetylation of Histone H3compared with single agent effects.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

What is claimed is:
 1. A method for treating lymphoma in a subject inneed thereof, the method comprising administering to the subject a HATactivator and a HDAC inhibitor, wherein the HAT activator is

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the HAT activator increases histone acetylation.
 3. The methodof claim 1, wherein the HDAC inhibitor increases histone acetylation. 4.The method of claim 2, wherein histone acetylation comprises acetylationof histones H2B, H3, H4, or a combination thereof.
 5. The method ofclaim 2, wherein histone acetylation comprises acetylation of histonelysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or acombination thereof.
 6. The method of claim 1, wherein the HAT activatorincreases p53 acetylation.
 7. The method of claim 1, wherein the HDACinhibitor increases p53 acetylation.
 8. The method of claim 1, whereinthe HDAC inhibitor is romidepsin, vorinostat, belinostat, panobinostat,entinostat, mocetinostat, abexinostat, quisinostat or gavinostat.
 9. Themethod of claim 1, wherein the HDAC inhibitor is romidepsin orvorinostat.
 10. The method of claim 1, wherein the HDAC inhibitor isromidepsin.
 11. The method of claim 1, wherein the HDAC inhibitor ischidamide, resminostat, givinostat, or kevetrin.
 12. The method of claim1, wherein the lymphoma is Hodgkin's lymphoma, non-Hodgkin's lymphoma, Bcell lymphoma, T cell lymphoma, or follicular lymphoma.
 13. The methodof claim 12, wherein the B cell lymphoma is diffuse large B-celllymphoma.
 14. The method of claim 13, wherein the diffuse large B-celllymphoma is a germinal center-derived diffuse large B cell lymphoma, anactivated B-cell-derived (ABC) diffuse large B-cell lymphoma, ornon-germinal center diffuse large B cell lymphoma.
 15. The method ofclaim 1, wherein the subject has at least one mutant HAT enzyme gene.16. The method of claim 1, wherein the subject has a wildtype EP300 andwildtype CREBBP gene.
 17. The method of claim 1, wherein the subject hasa wildtype EP300 and CREBBP mutant gene.
 18. The method of claim 17,wherein the mutation is on only one allele of EP300.
 19. The method ofclaim 1, wherein the subject has a mutant EP300 and CREBBP wildtypegene.
 20. The method of claim 19, wherein the mutation is on only oneallele of EP300.
 21. The method of claim 1, wherein the subject has amutant EP300 and CREBBP mutant gene.
 22. The method of claim 21, whereinthe mutation is on only one allele of EP300 and on only one allele ofCREBBP.
 23. The method of claim 1, wherein the HAT activator and a HDACinhibitor are administered separately.
 24. The method of claim 23,wherein the HAT activator and a HDAC inhibitor are administered atdifferent times.